TW201522374A - Anti-CRTh2 antibodies and methods of use - Google Patents

Abstract

The invention provides anti-CRTh2 antibodies and methods of using the same.

Description

Anti-CRTH2 antibody and method of use Cross-reference to related applications

The present application claims priority to U.S. Provisional Application Serial No. 61/786,370, the entire disclosure of which is hereby incorporated by reference.

Submission of sequence listing on ASCII text file

The contents of the following ASCII text file are incorporated by reference in their entirety: Computer-readable form (CRF) sequence listing (file name: 146392017340SEQLIST.TXT, record date: March 6, 2014, size: 115KB).

The present invention relates to anti-CRTh2 antibodies and methods of use thereof.

The chemoattractant receptor homolog (CRTh2), which is expressed on T helper 2 cells, is a member of the G-protein coupled receptor (GPCR) family. CRTh2 regulates the chemotaxis of eosinophils, basophilic spheres, and T helper type 2 (Th2) cells in response to prostaglandin D2 (PGD2). It has long been believed that these cell types, particularly Th2 cells, cause the pathogenesis of allergic diseases such as asthma. It has been shown in animal models that CRTh2 inhibition leads to attenuated airway hyperresponsiveness and inflammation. Lukacs et al; Am. J. Phsiol. Lung Cell. Mol. Physiol. 295: L767-779, 2008. For example, ramatroban, a dual-lipidin A2 receptor and CRTh2 receptor antagonist, suppresses eosinophil chemotaxis in vitro and in vivo and has been approved for use in Japan. Treat allergic rhinitis. Bosniak, B, et al., Respiratory Research 12: 114, 2011. Many other CRTh2 antagonists, such as 4-aminotetrahydroquinoline derivatives or indoleacetic acid derivatives, are currently under development. Pettipher; Br. J. Pharmacol. 153 (Suppl 1): S191-199, 2008; Royer et al; Eur. J. Clin. Invest. 38: 663-671, 2008; Stebbins et al; Eur. J. Pharmacol. 638: 142-149, 2010.

The invention provides anti-CRTh2 antibodies and methods of use thereof.

In one aspect, provided herein are isolated antibodies that bind to human CRTh2 and consume CRTh2 expressing cells when administered in a therapeutically effective amount to a human subject. In some embodiments, the anti-CRTh2 anti-system is engineered. In some embodiments, an anti-CRTh2 anti-system is produced by recombinant methods (eg, by transfecting or transforming a host cell with one or more nucleic acids encoding the antibody in vitro (eg, in cell culture)). In some embodiments, the host cell is a prokaryotic cell (eg, a bacterial cell) or a eukaryotic cell (eg, a CHO cell, a lymphoid cell).

In some embodiments, the antibody consumes one or more of the following types of CRTh2 expressing cells: Th2 cells, mast cells, eosinophils, basophilic spheres, or congenital type 2 (IT2) cells. In some embodiments, the antibody is engineered to improve ADCC and/or CDC activity. In some embodiments, the antibody is engineered to improve ADCC and/or reduce CDC activity. In some embodiments, the antibody is afucosylated. In some embodiments, the anti-system is produced in a cell line having an alpha 1,6-fucosyltransferase (Fut8) knockout. In some embodiments, the anti-system is produced in a cell line that overexpresses beta 1,4- N -ethyl glucosamine transferase III (GnT-III). In some embodiments, the cell line additionally overexpresses Golgi alpha-mannosidase II (ManII). In some embodiments, the antibody comprises at least one amino acid substitution that improves ADCC and/or CDC activity in the Fc region. In some embodiments, the amino acid is substituted for S298A/E333A/K334A.

In some embodiments, the anti-system naked antibody. In some embodiments, the anti-system embedding Combine antibodies. In some embodiments, the anti-system humanizes the antibody. In one embodiment, the anti-systematic human antibody. In some embodiments, the anti-system bispecific antibody. In some embodiments, the anti-system IgG1 antibody.

In some embodiments, the antibody binds to CRTh2 of a non-human primate. In some embodiments, the antibody binds to rhesus monkey and/or cynomolgus CRTh2.

In some embodiments, the antibody competitively inhibits binding of at least one of the following antibodies to human CRTh2: 19A2, 8B1, 31A5, 3C12, and any of the humanized antibodies described herein. In some embodiments, ELISA assays are used to determine competitive binding. In some embodiments, the antibody binds to an epitope of human CRTh2 that is identical or overlaps with a CRTh2 epitope that binds to at least one of the following anti-CRTh2 antibodies: 19A2, 8B1, 31A5, 3C12, and any of Humanized antibodies. In some embodiments, the antibody comprises six hypervariable regions (HVR) from one of the following anti-CRTh2 antibodies: 19A2, 8B1, 31A5, 3C12, and any of the humanized antibodies described herein.

In some embodiments, the antibody further blocks CRTh2 signaling. In some embodiments, the antibody prevents CRTh2 from expressing cells in response to recruitment of prostaglandin D2. In some embodiments, the antibody blocks the Ca2 ⁺ flux in the CRTh2 expressing cells. In some embodiments, the antibody binds to human CRTh2 with a Kd value of about 100 nM or less.

In some embodiments, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and X ₁ ISNGGSTTX ₂ YPGTVEG (SEQ ID NO :5) of HVR-H2, wherein X ₁ is Y or R, and X ₂ is Y or D. In some embodiments, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35 or 36, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and comprising SEQ ID NO: 32 or 33 HVR-H2 of the amino acid sequence. In some embodiments, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28, and the amino acid comprising SEQ ID NO: Sequence of HVR-H2.

In another aspect, provided herein are isolated anti-CRTh2 antibodies comprising a light chain and a heavy chain variable region, wherein the light and heavy chain variable regions comprise six hypervariable region (HVR) sequences: (i) HVR-L1 comprising RASENIYXNLA (SEQ ID NO: 1), wherein X is S, W or Y; (ii) HVR-L2 comprising AATQLAX (SEQ ID NO: 2), wherein X is D, E or S; Iii) HVR-L3 comprising QHFWITPWT (SEQ ID NO: 3); (iv) HVR-H1 comprising X ₁ YX ₂ MS (SEQ ID NO: 4), wherein X ₁ is S or F and X ₂ is S , L or K; (v) HVR-H2 comprising X ₁ ISNGGSTTX ₂ YPGTVEG (SEQ ID NO: 5), wherein X ₁ is Y or R, and X ₂ is Y or D; and (vi) comprises HRTNWDFDY (SEQ ID NO: 6) HVR-H3.

In another aspect, provided herein is an isolated anti-CRTh2 antibody comprising a light chain and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence comprising SEQ ID NO: 7, 8, or 9. HVR-L1, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, 11 or 12 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody further comprises a heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16 or 17, comprising SEQ ID NO: 18, 19 HVR-H2 of the amino acid sequence of 20 or 21 and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6.

In another aspect, provided herein is an isolated anti-CRTh2 antibody comprising a light chain and a heavy chain variable region comprising a heavy chain variable region comprising SEQ ID NOs: 13, HVR-H1 of the amino acid sequence of 15, 16 or 17, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, 19, 20 or 21 and HVR comprising the amino acid sequence of SEQ ID NO: 6. -H3.

In some embodiments, the antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (v) HVR-H2 comprising SEQ ID NO: an amino acid sequence of 19; and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15; (v) HVR-H2 comprising SEQ ID NO: amino acid sequence of 20; and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6.

In another aspect, provided herein is an isolated anti-CRTh2 antibody comprising a light chain and a heavy chain variable region, wherein the light chain variable region comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In another aspect, provided herein is an isolated anti-CRTh2 antibody comprising a light chain and a heavy chain variable region comprising a heavy chain variable region comprising: an HVR comprising the amino acid sequence of SEQ ID NO: -H1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20 and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15; (v) HVR-H2 comprising SEQ ID NO: amino acid sequence of 20; and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6.

In another aspect, provided herein is an isolated anti-CRTh2 antibody comprising a light chain and a heavy chain variable region, wherein the antibody comprises a VL sequence selected from the group consisting of SEQ ID NOs: 38-53. In some embodiments, the antibody further comprises a VH sequence selected from the group consisting of SEQ ID NOs: 54-65. In another aspect, provided herein is an isolated anti-CRTh2 antibody comprising a light chain and a heavy chain variable region, wherein the antibody comprises an antibody selected from the group consisting of SEQ ID NO: 54-65 The VH sequence of the group. In some embodiments, provided herein are isolated anti-CRTh2 antibodies comprising a light chain variable region comprising a VL sequence selected from the group consisting of SEQ ID NOs: 38-48 and comprising selected from the group consisting of SEQ ID NOs: 54-60 The heavy chain variable region of the VH sequence of the group.

In some embodiments, the antibody comprises the VL sequence of SEQ ID NO: 40 and the VH sequence of SEQ ID NO: 57. In some embodiments, the antibody comprises the VL sequence of SEQ ID NO:39 and the VH sequence of SEQ ID NO:55. In some embodiments, the antibody comprises the VL sequence of SEQ ID NO: 41 and the VH sequence of SEQ ID NO: 57.

In some embodiments, the anti-system monoclonal antibody. In some embodiments, the anti-systematic antibody or chimeric antibody. In some embodiments, at least a portion of the framework sequences of the antibodies are human consensus framework sequences. In some embodiments, the anti-system is selected from the group consisting of Fab, Fab'-SH, Fv, scFc or (Fab') ₂ fragments.

In another aspect, provided herein is an isolated nucleic acid encoded by any of the antibodies described herein. In another aspect, provided herein is a host cell comprising a nucleic acid described herein. In another aspect, provided herein is a method of producing an antibody comprising culturing a host cell to produce an antibody. In some embodiments, the method further comprises recovering the antibody produced by the host cell.

In another aspect, provided herein is an immunoconjugate comprising any of the antibodies and cytotoxic agents described herein. In some embodiments, the immunoconjugate is in a pharmaceutical composition. Immunoconjugates can be used in any of the methods described herein.

In another aspect, provided herein is a pharmaceutical composition comprising any of the anti-CRTh2 antibodies described herein and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a method of treating asthma comprising administering an effective amount of an anti-CRTh2 antibody to an individual, wherein the antibody depletes CRTh2 expressing cells in the individual.

In some embodiments, the antibody consumes one of the following types of CRTh2 expressing cells or Many: Th2 cells, mast cells, eosinophils, basophils or congenital type 2 (IT2) cells. In some embodiments, the anti-CRTh2 antibody is derived from a lung tissue that depletes CRTh2 expressing cells. In some embodiments, the anti-CRTh2 antibody consumes CRTh2 expressing cells from bronchoalveolar lavage fluid. In some embodiments, the anti-CRTh2 antibody consumes at least 50% of at least one type of CRTh2 expressing cells from the lung compared to the baseline prior to administration of the antibody. In some embodiments, the anti-CRTh2 antibody consumes at least 80% of at least one type of CRTh2 expressing cells from the lung compared to the baseline prior to administration of the antibody. In some embodiments, the anti-CRTh2 antibody consumes at least 90% of at least one type of CRTh2 expressing cells from the lung compared to the baseline prior to administration of the antibody. In some embodiments, the individual has pauci granulocytic asthma. In some embodiments, the amount of one or more cytokines in the individual is reduced following administration of the anti-CRTh2 antibody. In some embodiments, reducing the amount of one or more cytokines produced by at least one of the following cell types: Th2 cells, mast cells, eosinophils, basophilic spheres, or congenital type 2 (IT2) cells . In some embodiments, the amount of one or more of IL-4, IL-5, IL-9, IL-13, IL-17, histamine or leukotriene in the individual is reduced. In some embodiments, the individual has an inhaled corticosteroid, a short acting beta 2 agonist, a long acting beta 2 agonist, or a combination thereof that controls inadequate asthma. In some embodiments, the individual is a human. In some embodiments, the anti-CRTh2 anti-system is an antibody described herein.

In another aspect, provided herein is a method of treating a condition mediated by a CRTh2 expressing cell, comprising administering an effective amount of an anti-CRTh2 antibody to an individual, wherein the antibody depletes a CRTh2 expressing cell in the individual.

In some embodiments, the condition is selected from the group consisting of asthma, granule-deficient asthma, atopic dermatitis, allergic rhinitis, acute or chronic airway hypersensitivity, eosinophilia syndrome, eosinophilia Cystic esophagitis, Churg-Strauss syndrome, idiopathic pulmonary fibrosis, inflammation associated with cytokines, inflammation associated with CRTh2 expressing cells, and inflammation associated with CRTh2 expressing cells Sexual neoplasms, chronic idiopathic urticaria, chronic spontaneous urticaria, physical urticaria, cold urticaria, stress urticaria, bullous pemphigoid, nasal sputum, food allergies and allergic bronchopulmonary Ascariasis (ABPA). In some embodiments, the anti-CRTh2 antibody is an antibody described herein.

In another aspect, provided herein is a method of reducing the amount of a cytokine in an individual comprising administering an effective amount of an anti-CRTh2 antibody to the individual, wherein the antibody depletes the CRTh2 expressing cell in the individual. In some embodiments, the amount of one or more of IL-4, IL-5, IL-9, IL-13, IL-17, histamine, or leukotriene in the individual is reduced. In some embodiments, the anti-CRTh2 antibody is an antibody described herein.

It will be understood that one, some or all of the properties of the various embodiments described herein may be combined to form other embodiments of the invention. These and other aspects of the invention will be apparent to those skilled in the art. These and other embodiments of the invention are further illustrated by the following detailed description.

Figure 1 shows that the CRTh2 line is expressed on human "Th2" molecular cells. CRTh2 expression was assessed by flow cytometry using human PBMC population or anti-human CRTh2 Ab (BM16) on cultured human cells, as indicated.

Figure 2 shows that CRTh2+ memory CD4+ T cells produce more than 95% of memory CD4+ T cell Th2 cytokines (I1-4, IL-5, IL13 and IL-9) when compared to CRTh2-memory CD4+ T cells. CRTh2+CD45RO+ and CRTh2-CD45RO+ memory CD4+ T cells were isolated from human PBMC by flow cytometry and stimulated with anti-CD3 and anti-CD28 antibodies for 48 hours at 37 °C. The supernatant was collected and quantified as shown by Luminex.

Figures 3A-F show the reactivity of mouse or humanized anti-CRTh2 antibodies by flow cytometry for CRTh2 expressed on cell lines or with primary basophilic and eosinophils. Figure 3A shows the reactivity of mouse anti-CRTh2 fusion tumor antibodies (pure lines 19A2, 8B1, 31A5 and 3C12) compared to control Ab (20 ug/ml, color histogram), This was carried out by flow cytometry on human, rhesus or cynomolgus CRTh2 expressed on 293 cells and wild type 293 cells not expressing CRTh2. The primary antibody concentration used was 20 ug/ml (black line), 2 ug/ml (grey line) and 0.2 ug/ml (light gray line). Figure 3B shows mouse anti-CRTh2 antibody (19A2 and 8B1 cloned using mIgG2a) and human, rhesus monkeys expressed on 300.19 cells by flow cytometry compared to isotype control Ab (color histogram) The reactivity of CRTh2, which is flagged by the amino terminal of cynomolgus monkey, and wild type 300.19 cells which do not express CRTh2. The primary antibody concentration used was 1 ug/ml (human, cynomolgus; black line) or 5 ug/ml (rhesus, wild type; black line) and 0.5 ug/ml (rhesus, wild type; gray line). The anti-Flag Ab was used at 0.7 ug/ml. Figure 3C shows the reactivity of mouse anti-human CRTh2 antibodies (19A2, 8B1, 31A5, 3C12) to basophilic and eosinophils on human PBMC by flow cytometry. PBMC with anti-CRTh2 antibody at 5 ug/ml (black line), 0.5 ug/ml (grey line) or with isotype control Ab at 5 ug/ml (light gray line), 0.5 ug/ml (color histogram), followed Fluorescently labeled secondary anti-mouse IgG, anti-CD16, anti-HLADR and anti-CD123 were incubated together. Figure 3D and Figure 3E show that humanized h19A2.v1 and engineered humanized h19A2.v12 anti-CRTh2 antibodies were compared to 293 cells (Fig. 3D) or 300.19, respectively, compared to wild type 293 or 300.19 cells that did not express CRTh2, respectively. The reactivity of the amino-terminal gD-labeled or flag-tagged human, rhesus or cynomolgus CRTh2 expressed on cells (Fig. 3E). The primary anti-CRTh2 Ab concentration used was as follows: 10 ug/ml (black line), 1 ug/ml (grey line) and 0.1 ug/ml (light gray line); isotype control Ab (2H7, color histogram) was 10 ug/ For ml use, the anti-gD anti-system was used at 2 ug/ml and the anti-Flag Ab was used at 0.7 ug/ml. Figure 3F shows that anti-CRTh2 antibodies h19A2.v1 and h19A2.v12 at 10 ug/ml (black line) against primary human, cynomolgus and rhesus basophils from peripheral blood and primary human eosinophils and isoforms Control Ab (color histogram) compared to FACS binding.

4A-B show Scatchard analysis of the binding affinity of anti-CRTh2 antibody (mIgG or hFab) for CRTh2 on 293 cells or 300.19 cells. Figure 4A shows radioligand cell binding assays of human anti-CRTh2 whole antibodies 19A2 and 8B1 for human CRTh2 expressed on 293 cells or 300.19 cells, as indicated. Figure 4B shows the radioligand cell binding assay of humanized h19A2.v12 or h19A2.v60 Fab fragments for human or cynomolgus CRTh2 expressed on 293 cells. The dissociation constant (K _D ) against CRTh2 Ab is indicated in the graph. The binding/total indicates the concentration ratio of the ¹²⁵ I-labeled antibody to the total antibody; the overall indicates the concentration of the ¹²⁵ I-labeled and unlabeled antibody.

Figure 5 shows that anti-CRTh2 antibodies 8B1 and 3C12 prevent PGD2-induced calcium mobilization. Calcium flux in response to PGD2 stimulation was monitored by flow cytometry in the presence of anti-CRTh2 or isotype control antibodies in a subset of Th2 cells (CD4+CCR4+CCR6-CXCR3-) from in vitro polarized Th2 cells. The CRTh2 receptor antagonist CAY10471 was included as a positive control.

Figures 6A-B show the involvement and characteristics of human CRTh2 BAC transgenic mice. Figure 6A depicts a 171 kb genomic region containing the human CRTh2 gene on chromosome 11 which was introduced into C57BL/6 mice to generate hCRTh2 BAC transgenic mice. Figure 6B shows blood basophilic bulbs (CD123+FceRI+), blood eosinophils (CCR3+), peritoneal mast cells (FceRI+CD117+), axillary lymph nodes in hCRTh2.Bac.Tg cell line 85 by flow cytometry. CD4+CD44hi T cells (induced by the Th2 polarizer papain) and mesenteric lymph node congenital T helper 2 cells (Lin-CD117+ intensively injected with mouse IL-17E plasmid by tail vein) showed human CRTh2 expression (antibody BM16) ). For comparison, flow cytometry analysis of human CRTh2 expressed on human cells is shown. Basophilic globules, eosinophils, and IT2 cells from PBMCs, mast cells from human bone marrow-derived mast cells, and Th2 cells (CCR4+CXCR3-) are isolated from human PBMCs under Th2 polarization. T cell differentiation.

Figures 7A-B show that anti-CRTh2 antibodies consume blood basophilic and eosinophils in vivo in human CRTh2.Bac.Tg mice. Determination of CRTh2+ basophilic ball in blood by flow cytometry on day -4 (Fig. 7A) or 4 hours (Fig. 7B) before treatment with anti-CRTh2 Ab (19A2, 3C12 or 8B1, as indicated) +FceRI+) and eosinophilic ball (CCR3+) The number of baselines. Human CRTh2.Bac.Tg mice were treated with anti-CRTh2 or isotype control antibody at 200 ug/mouse i.v. (Fig. 7A) or 150 ug/mouse i.v. (Fig. 7B). Blood basophilic bulbs and eosinophilic bulb consumption were assessed by flow cytometry on day 3, day 6, or day 7, as indicated. The % consumption of anti-CRTh2 compared to the anti-Swine grass isotype control antibody is indicated in Figure 7B.

8A-B shows that anti-CRTh2 antibody 19A2 treatment consumes innate immune cells and reduces Th2 bronchoalveolar lavage (BAL) cytokine production in a TNP-OVA-induced chronic asthma model in CRTh2.Bac.Tg mice. Figure 8A shows the number of basophilic, eosinophilic, and mast cells assessed by flow cytometry in lung tissue by Boundary Cell Count and Flow Cytometry in BAL (Figure 8A). The % consumption of anti-CRTh2 compared to the anti-Swine grass isotype control antibody is indicated in the graph. Figure 8B shows the concentration of IL-4 and IL-13 as determined by ELISA in BAL. The % decrease compared to the isotype control antibody by anti-CRTh2 treatment is indicated in the graph.

Figure 9A-B shows that anti-CRTh2 antibody 19A2 depletes human-produced IL-4 in Th2 cells or congenital helper 2 (IT2) cells in human CRTh2.Bac.Tg mice in SCID mice. Figure 9A: Transfer of in vitro polarized human Th2 cells from PBMC into SCID mice and further in vivo by injection of rhIL-4 plus anti-IFN-g and anti-IL-12 mAb in afucosylation resistance Polarization in the presence of CRTh2 19A2 antibody or isotype control antibody for 7 days. After 7 days, the percentage of CD4 T cells producing IL-4 or IFN-g was determined. For this purpose, spleen cells were starved and stimulated ex vivo with PdBu (50 ng/mL) and ionomycin (500 ng/mL) for 4.5 hours with brefeldin A added during the last 3 hours of stimulation. , BFA). Cells were surface-stained with anti-hCD4 and lineage cells were stained with anti-mCD45, anti-mTer119 and anti-hCD19; cells were fixed and stained with anti-hIFNg and anti-hIL-4 to detect cytokine positive cells. Figure 9B: Human CRTh2.Bac.Tg mice were injected with 50 ug of mouse IL-17E encoding plasmid followed by anti-CRTh2 or isotype control Ab. Percentage of IT2 cells in the mesenteric lymph nodes by flow cytometry on day 3 after treatment And the total number. The % consumption of anti-CRTh2 compared to the anti-Swine grass isotype control antibody is indicated in the graph.

Figure 10 shows the amino acid sequence of the murine anti-CRTh2 antibody 19A2 light chain (SEQ ID NO: 49) and heavy chain (SEQ ID NO: 61) variable region. Kabat CDRs, Chothia CDRs and Contact CDR sequences of heavy and light chains are provided.

Figure 11A-B shows an amino acid sequence alignment of the light and heavy chain variable regions of a humanized anti-CRTh2 antibody derived from antibody 19A2. Figure 11A shows a light chain variable region sequence alignment. Providing the light chain Kabat CDR, Chothia CDR and Contact CDR sequences of each antibody (hu19A2.v1 (SEQ ID NO: 38), hu19A2.v12 (SEQ ID NO: 39), hu19A2.v46 (SEQ ID NO: 39), Hu19A2.v52 (SEQ ID NO: 40), hu19A2.v58 (SEQ ID NO: 42), hu19A2.v60 (SEQ ID NO: 41), hu19A2.v61 (SEQ ID NO: 42), hu19A2.v62 (SEQ ID NO: 41), hu19A2.v63 (SEQ ID NO: 43), hu19A2.v64 (SEQ ID NO: 42), hu19A2.v65 (SEQ ID NO: 43), hu19A2.v66 (SEQ ID NO: 44), hu19A2 .v67 (SEQ ID NO: 45), hu19A2.v68 (SEQ ID NO: 44), hu19A2.v69 (SEQ ID NO: 45), hu19A2.v70 (SEQ ID NO: 46), hu19A2.v71 (SEQ ID NO) :47), hu19A2.v72 (SEQ ID NO: 48)). Figure 11B shows the heavy chain variable region sequence alignment. Providing the heavy chain Kabat CDR, Chothia CDR and Contact CDR sequences of each antibody (hu19A2.v1 (SEQ ID NO: 54), hu19A2.v12 (SEQ ID NO: 55), hu19A2.v46 (SEQ ID NO: 57), Hu19A2.v52 (SEQ ID NO: 57), hu19A2.v58 (SEQ ID NO: 57), hu19A2.v60 (SEQ ID NO: 57), hu19A2.v61 (SEQ ID NO: 55), hu19A2.v62 (SEQ ID NO: 55), hu19A2.v63 (SEQ ID NO: 55), hu19A2.v64 (SEQ ID NO: 60), hu19A2.v65 (SEQ ID NO: 60), hu19A2.v66 (SEQ ID NO: 55), hu19A2 .v67 (SEQ ID NO: 55), hu19A2.v68 (SEQ ID NO: 60), hu19A2.v69 (SEQ ID NO: 60), hu19A2.v70 (SEQ ID NO: 54), hu19A2.v71 (SEQ ID NO: 54), hu19A2.v72 (SEQ ID NO: 54)).

Figure 12 shows an amino acid sequence alignment of murine anti-CRTh2 antibody 8B1 with 3C12 and humanized anti-CRTh2 hu8B1.v1 light chain and heavy chain variable regions (mu8B1-light chain variable region (SEQ ID NO: 50) , mu8B1-heavy chain variable region (SEQ ID NO: 62); mu3C12-light chain variable region (SEQ ID NO: 51), mu3C12-heavy chain variable region (SEQ ID NO: 63); hu8B1.v1 a light chain variable region (SEQ ID NO: 52), hu8B1.v1 - heavy chain variable region (SEQ ID NO: 64)). The light and heavy chain Kabat CDRs, Chothia CDRs and Contact CDR sequences of each antibody are provided.

Figure 13 shows the amino acid sequence of murine anti-CRTh2 antibody 31A5. The light and heavy chain Kabat CDRs, Chothia CDRs and the Contact CDR sequences of the antibody 31A5 (mu31A5-light chain variable sequence (SEQ ID NO: 53), mu31A5-heavy chain variable sequence (SEQ ID NO: 65)) are provided.

Figure 14 shows an amino acid sequence alignment of the light chain and heavy chain variable regions of the humanized anti-CRTh2 antibodies hu19A2.v1 and hu19A2.v52. Figure 14A shows a light chain variable region sequence alignment (hu19A2.vl - light chain variable region (SEQ ID NO: 38); hu19A2.v52 - light chain variable region (SEQ ID NO: 40)). The light chain Kabat CDR, Chothia CDR and Contact CDR sequences of each antibody are provided. Figure 14B shows alignment of heavy chain variable region sequences (hu19A2.vl-heavy chain variable region (SEQ ID NO: 54); hu19A2.v52-heavy chain variable region (SEQ ID NO: 57)). The heavy chain Kabat CDR, Chothia CDR and Contact CDR sequences of each antibody are provided.

Figures 15A-C show the reactivity of humanized and humanized affinity matured anti-CRTh2 antibodies to CRTh2 or primary basophilic and eosinophils expressed on cell lines by flow cytometry. Figure 15A shows anti-CRTh2 antibody by flow cytometry 19A2 humanization (h19A2.v1) and humanized affinity maturation (h19A2.v46, h19A2.v52) at 1 ug/ml (black line) and 0.1 ug/ml (grey line) ) compared to the control Ab (at 1 ug/ml, color histogram) The reactivity of human CRTh2 expressed on 293 cells and wild type 293 cells not expressing CRTh2. Figure 15B shows humanized and humanized affinity matured 19A2 anti-CRTh2 antibodies (h19A2.v1, h19A2.v12, h19A2.V46, h19A2.v52) and control Ab by flow cytometry (at 0.55 ug/ml, color histogram) Figure) Reactivity compared to human, cynomolgus or rhesus CRTh2 expressed on 293 cells and wild type 293 cells not expressing CRTh2. The primary antibody concentration used was 0.55 ug/ml (black line), 0.18 ug/ml (very dark gray line), 0.06 ug/ml (dark gray line), 0.02 ug/ml (grey line), and 0.006 ug/ml (shallow). Gray line). Figure 15C shows the reactivity of humanized affinity anti-human CRTh2 antibody h19A2.v52 to basophilic and eosinophils on human, cynomolgus or rhesus PBMC by flow cytometry. PBMC together with fluorescently labeled anti-CRTh2 antibody at 15 ug/ml (black line), 5 ug/ml (very dark gray line), 1.7 ug/ml (dark gray line), 0.6 ug/ml (grey line) or 0.2 Ug/ml (light gray line) or in combination with an isotype control Ab at 15 ug/ml (color histogram) in combination with lineage-specific antibodies to detect basophilic and eosinophils as described in Materials and Methods .

Figure 16 shows the Scatchard analysis of the binding affinity of anti-CRTh2 antibody (Fab fragment) to CRTh2 on the surface of 293 cells. Figure 16A-B shows homologous competition radioligand cell binding assays of humanized h19A2.v52 Fab fragments for human or cynomolgus CRTh2 expressed on 293 cells. Figure 16C-D shows homologous competition radioligand cell binding assays of humanized h19A2.v46 Fab fragments for human or cynomolgus CRTh2 expressed on 293 cells. The dissociation constant (K _D ) against CRTh2 Ab is indicated in the graph. Binding/overall indicates the ratio of antibodies bound to ¹²⁵ I and total ¹²⁵ I labeled antibodies used in each assay.

17A-B show the effect of anti-CRTh2 antibody on forskolin-induced PGD2-mediated inhibition of cAMP content or on forskolin-induced cAMP content in 293 cells expressing human CRTh2. Figure 17A shows that anti-CRTh2 antibody h19A2.v52 does not affect PGS2-mediated inhibition of forskolin-induced cAMP content in 293 cells expressing human CRTh2. In contrast, humanized h8B1 antibody blocked forsk in a dose-dependent manner PGD2-mediated inhibition of forest-induced cAMP content. Figure 17B shows that anti-CRTh2 antibodies h8B1 and h19A2.v52 did not affect forskolin-induced cAMP levels in the absence of PGD2 in 293 cells expressing human CRTh2. In contrast, ligand PGD2 reduced forskolin-induced cAMP content in a dose-dependent manner.

18A-C show that murine anti-CRTh2 antibody 19A2 (mIgG2a) consumes basophilic globules and eosinophils in blood, spleen and bone marrow of human CRTh2.Bac.Tg mice in vivo. The baseline number of CRTh2+ basophilic (CD123+FceRI+) and eosinophilic (CCR3+) blood was determined by flow cytometry on day -7 before treatment with anti-CRTh2 Ab 19A2 (Fig. 18A and Fig. 18B). As indicated. Human CRTh2.Bac.Tg mice were treated with anti-CRTh2 or isotype control antibody at 20 ug/mouse or 100 ug/mouse i.v. Evaluation of basophilic spheres by flow cytometry as indicated on day 3 of the third day in the blood (Figures 18A and 18B) or on day 7 in the spleen and bone marrow (BM) (Figure 18C) Eosinophilic ball consumption. The symbols represent data from individual mice.

Figure 19A-C shows dose response of basophilic or eosinophilic depletion in blood, spleen and bone marrow of human CRTh2.Bac.Tg mice after a single dose of humanized anti-CRTh2 antibody h19A2.v52 (hIgG1) And duration. The baseline number of CRTh2+ eosinophilic spheres (CCR3+) in the blood was determined by flow cytometry on day -3 before treatment with h19A2.v52 (Fig. 19A), as indicated. Human CRTh2.Bac.Tg mice were treated with anti-CRTh2 or isotype control antibody at 10 ug/mouse or 200 ug/mouse i.v. The basophilic ball and eosinophilic consumption in blood (Fig. 19A), spleen (Fig. 19B) and bone marrow (BM) (Fig. 19C) were evaluated by flow cytometry on days 2, 7, and 14. As indicated. The symbols represent data from individual mice.

Figure 20A-B shows the depleting anti-CRTh2 19A2 mIgG2a antibody with effector function in the TNP-OVA-induced chronic asthma model in the TNP-OVA-induced chronic asthma model with congenital immune cell depletion and decreased Th2 BAL cytokine production. It is more potent than the non-consuming anti-CRTh2 19A2 mIgG2a_DANA Fc mutant antibody. Figure 20A shows the lung group The number of basophilic, eosinophilic, and mast cells assessed by flow cytometry and in BAL by combinatorial cell counting and flow cytometry in the BAL (Fig. 20A). The % consumption of the anti-CRTh2 19A2 mIgG2a antibody and the Fc mutant 19A2 mIgG2a_DANA antibody compared to the anti-Swine grass isotype control antibody is indicated in the graph. Figure 20B shows the concentration of IL-4 in BAL as determined by ELISA. The % decrease in anti-CRTh2 19A2 mIgG2a antibody and Fc mutant 19A2 mIgG2a_DANA antibody compared to the anti-Swine grass isotype control antibody is indicated in the graph.

I. Definition

For the purposes herein, "receptor human framework" is an amino acid comprising a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. The framework of the sequence, as defined below. The acceptor human framework "derived from" the human immunoglobulin framework or the human consensus framework may comprise the same amino acid sequence, or it may contain amino acid sequence changes. In some embodiments, the amount of amino acid change is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or Smaller or 2 or smaller. In some embodiments, the sequence of the VL receptor human framework is identical to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"Affinity" refers to the sum of the intensities of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, an antibody and an antigen). The affinity of the molecule X for its partner Y is generally represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are set forth below.

An "affinity mature" anti-system refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have an alteration, such alterations that result in improved resistance The affinity of the body for the antigen.

The term "CRTh2" as used herein, unless otherwise indicated, refers to from any vertebrate (eg, primates (eg, humans, rhesus monkeys, cynomolgus CRTh2) and rodents (eg, mice and rats) )) Any of the natural CRTh2. The term encompasses "full length" untreated CRTh2 and any form of CRTh2 produced by the treatment of cells. The term also encompasses naturally occurring variants of CRTh2, for example, splice variants or dual gene variants. An amino acid sequence of an exemplary human CRTh2 is shown in SEQ ID NO:84. An amino acid sequence of an exemplary rhesus monkey CRTh2 is shown in SEQ ID NO:85. The amino acid sequence of an exemplary cynomolgus CRTh2 is shown in SEQ ID NO:86. See, for example, L. Cosmi et al., Eur. J. Immunol. 30(10): 2972-9 (2000); K. Nagat et al., FEBS Lett. 459(2): 195-9 (1999); and K. Nagata et al., J. Immunol. 162(3): 1278-86 (1999).

Human CRTH2 sequence (SEQ ID NO: 84)

Rhesus CRTH2 sequence (NCBI reference number XM_001084746) (SEQ ID NO: 85)

Cynomolgus CRTH2 sequence (SEQ ID NO: 86)

The terms "anti-CRTh2 antibody" and "antibody that binds to CRTh2" refer to an antibody that binds CRTh2 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent for targeting CRTh2. In one embodiment, the degree of binding of an anti-CRTh2 antibody to an unrelated non-CRTh2 protein is about 10% lower than the binding of the antibody to CRTh2, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of an antibody that binds to CRTh2 1μM, 100nM, 10nM, 1nM, 0.1nM, 0.01nM or 0.001 nM (eg 10 ^-8 M or less, such as 10 ^-8 M to 10 ^-13 M, such as 10 ^-9 M to 10 ^-13 M). In certain embodiments, the anti-CRTh2 antibody binds to a CRTh2 epitope that is conserved in CRTh2 from a different species.

The term "antibody" is used herein in its broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, as long as It exhibits the desired antigen binding activity.

"Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , bispecific antibodies; linear antibodies; single-chain antibody molecules (eg, scFv); The multispecific antibody formed by the fragment.

"Antibody binding to the same epitope" (as a reference antibody) refers to an antibody that blocks the binding of a reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody is used in a competitive assay. Binding to its antigen blocks 50% or more. An example competitive analysis is provided herein.

The term "chimeric" anti-system refers to an antibody from a particular source or species that is partially derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "type" of an antibody refers to the type of constant domain or constant region that the heavy chain has. There are five main types of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (eg, radioactive isotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ^{212 ,} and Lu); Chemotherapeutic agents or drugs (eg, methotrexate, adriamicin, vinca alkaloid (vincristine, vinblastine, etoposide), multiple Doxorubicin, melphalan, mitomycin C, chlorambucil, daunoruhicin or other intercalating agents; growth inhibitors; enzymes and Fragments thereof, such as lysozymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins from bacterial, fungal, plant or animal sources, including fragments and/or variants thereof; and various antitumor agents disclosed below Or an anticancer agent.

"Effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cell receptors) Bottom); and B cell activation.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the C-terminus of the Fc region may or may not be present in the amine acid (Lys447). Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, also known as the EU index, such as Kabat et al., Sequences of Proteins of Immunological Interest , 5th Edition, Public. Health Service, National Institutes of Health, Bethesda, MD, 1991.

"Framework" or "FR" refers to a variable domain residue other than a hypervariable region (HVR) residue. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences are typically found in the following sequences in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a native antibody or having a heavy chain comprising an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and derived therefrom Offspring, regardless of the number of passages. The nucleic acid content of the progeny and the parental cell may not be identical, but may contain mutations. Described herein are mutant progeny that have been screened or selected for initial transformed cells with the same function or biological activity.

A "human antibody" is an amino acid sequence having an amino acid sequence corresponding to an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody profile or other sequence encoding a human antibody. The definition of this human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

The "Human Common Framework" represents the framework of the most prevalent amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, subgroups of sequences are as subgroups of Kabat et al, Sequences of Proteins of Immunological Interest , 5th edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is a subgroup of κI as described by Kabat et al. (see above). In one embodiment, for VH, the subgroup is a subset of III as described by Kabat et al. (see above).

A "humanized" anti-system refers to a chimeric antibody comprising an amino acid residue from a non-human HVR and an amino acid residue from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the HVRs (eg, CDRs) correspond to non-human HVRs, and All or substantially all of the FR corresponds to the FR of the human antibody. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

The term "hypervariable region" or "HVR" as used herein, refers to a sequence in a region of an antibody variable domain that has a high degree of denaturation and/or forms a structurally defined loop ("hypervariable loop"). Typically, the native four-chain antibody comprises six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVR usually contains high-variation rings And/or amino acid residues from the "complementarity determining region" (CDR), which have the highest sequence variability and/or are involved in antigen recognition. The HVR region as used herein is comprised at positions 24-36 (for L1), 46-56 (for L2), 89-97 (for L3), 26-35B (for H1), 47-65 (for H2), and 93- Any number of residues within 102 (for H3). Thus, the HVR includes residues in the previously described positions:

A) 24-34 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

B) 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest , Version 5. Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

C) 30-36 (L1), 46-55 (L2), 89-96 (L3), 30-35 (H1), 47-58 (H2), 93-100a-j (H3) (MacCallum et al, J. Mol. Biol. 262: 732-745 (1996)).

In addition to CDR1 in VH, CDRs typically comprise an amino acid residue that forms a hypervariable loop. The CDR also contains a "specificity determining residue" or "SDR" which is a residue that contacts the antigen. The SDR is included in the region of the CDR called the abbreviated-CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are present in the amino acid residue L1 31-34, L-50-55, L3 89-96, H1 31-35B, H2 50-58 and H3 95-102. (See Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues (eg, FR residues) in the variable domain are herein based Kabat et al. numbered (see above).

An "immunoconjugate" is an antibody that is conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

"Individual" or "subject" is a mammal. mammal These include, but are not limited to, domestic animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates, such as monkeys), rabbits, and rodents (eg, Mouse and rat). In some embodiments, the individual (subject) or human is a human.

The "separated" anti-system is separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ions) Determined by exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al, J. Chromatogr. B 848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that typically contains a nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-CRTh2 antibody" refers to one or more nucleic acid molecules encoding an antibody heavy and light chain (or a fragment thereof) comprising a nucleic acid molecule in a single vector or a separate vector and present in The nucleic acid molecule at one or more positions in the host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homologous antibodies, ie, the individual antibodies comprising the population are identical and/or bind to the same epitope, with the exception of possible variant antibodies, eg, Natural mutations or variants produced during the production of a monoclonal antibody preparation, such variants are typically present in minor amounts. Each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen as compared to a multi-drug antibody preparation that typically includes different antibodies directed against different determinants (epitopes). Thus, the modifier "single plant" indicates that the antibody is characterized by a population of antibodies that are substantially homologous and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by a variety of techniques including, but not limited to, fusion knob methods, recombinant DNA methods, phage display methods, and utilization. Methods of transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods of making monoclonal antibodies are set forth herein.

"Naked antibody" refers to an antibody that has not been conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

"Native antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG anti-system is a heterotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C terminus, each heavy chain has a variable region (VH) (also known as a variable heavy chain domain or a heavy chain variable domain) followed by three constant domains (CH1, CH2 and CH3). Similarly, from N to C terminus, each light chain has a variable region (VL) (also known as a variable light chain domain or a light chain variable domain) followed by a constant light chain (CL) domain. The light chain of the antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain of the antibody.

The term "package insert" is used to refer to a specification that is typically included in a commercial package of a therapeutic product, containing information about the indication, usage, dosage, administration, combination therapy, contraindications, and/or warning about the use of the therapeutic product. .

The "amino acid sequence identity percentage (%)" relative to the reference peptide sequence is defined as the amino acid residue in the candidate sequence after aligning the sequence and introducing a gap (if necessary) to achieve the maximum sequence identity percentage. The percentage of amino acid residues in the reference peptide sequence is consistent and does not consider any conservative substitutions as part of sequence identity. For purposes of determining the percent identity of the amino acid sequence, the alignment can be accomplished in a variety of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). )software. Those skilled in the art will be able to determine appropriate parameters for the alignment sequence, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for the purposes of this document, the sequence comparison computer program ALIGN-2 was used to generate the value of the amino acid sequence identity %. ALIGN-2 sequence comparison computer The program was created by Genentech, and the source code and user files have been filed with U.S. Copyright Office, Washington D.C., 20559, which is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use in UNIX operating systems (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and are not changed.

In the case where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence of a given amino acid sequence A is given relative to (to), with or with a given amino acid sequence B. % identity (or % expressed as a given amino acid sequence A relative to, or with respect to, a certain amino acid sequence possessed or included for a given amino acid sequence B) is calculated as follows: 100 times Fraction X/Y, where X is the number of amino acid residues that are consistently matched by the sequence alignment program ALIGN-2 in the program alignment of A and B, and the total number of amino acid residues in Y system B . It will be appreciated that when the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the % identity of A with respect to the amino acid sequence of B will not be equal to the % identity of B with respect to the amino acid sequence of A. All amino acid sequence identity % values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph, unless explicitly stated otherwise.

The term "pharmaceutical formulation" refers to a formulation that is in a form that allows for the biological activity of the active ingredient contained therein to be effective and that does not contain other components that are unacceptable to the individual to which the formulation will be administered.

"Pharmaceutically acceptable carrier" means a component of a pharmaceutical formulation that is not toxic to the individual other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term "treatment" as used herein refers to a clinical intervention designed to alter the natural progression of a treated individual or treated cell during a course of clinical pathology. Desirable therapeutic effects include reducing the rate of disease progression, improving or ameliorating disease states, and alleviating or improving prognosis. In some embodiments, the treatment improves asthma control, reduces asthma exacerbations, improves lung function and/or Or improve the symptoms reported by the patient. For example, an individual is successfully "treated" if one or more of the symptoms associated with the condition are alleviated or eliminated.

"Combination" as used herein refers to administration of one form of treatment with another form of treatment. Similarly, "binding" refers to the administration of a form of treatment between, during, or after administration of another form of treatment to an individual.

The term "prevention" as used herein includes the prevention of the occurrence or recurrence of a disease in an individual. An individual may be susceptible to, susceptible to, or at risk of developing the condition, but the condition has not been diagnosed. In some embodiments, an anti-CRTh2 antibody described herein is used to delay the progression of a condition. In some embodiments, an anti-CRTh2 antibody described herein prevents asthma exacerbation and/or debilitates a non-functional or asthmatic state.

As used herein, an individual "at risk of developing a condition" may or may not have a detectable disease or condition of the disease, and may or may not exhibit a detectable disease or condition of the disease prior to the methods of treatment described herein. "At risk" means that the individual has one or more risk factors, which are measurable parameters associated with the development of the condition, as is known in the art. An individual having one or more of the risk factors has a higher probability of developing the condition than an individual who does not have one or more of the risk factors.

By "effective amount" is meant an amount effective to achieve a desired or indicative effect (including a treatment or preventative effect) at a desired dose for at least the desired period of time. An effective amount can be provided by one or more administrations.

A "therapeutically effective amount" is the minimum concentration required to achieve at least a measurable improvement of a particular condition. The therapeutically effective amount herein may vary depending on factors such as the disease state, age, sex and weight of the patient and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which the therapeutically beneficial effects of the antibody outweigh any toxic or detrimental effects thereof. "Prophylactically effective amount" means an amount effective to achieve the desired prophylactic effect at the desired dose over a desired period of time. Typically, but not necessarily, since the prophylactic dose is administered to the individual prior to or prior to the onset of the disease, the prophylactically effective amount can be less than the therapeutically effective amount.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of the native antibody (VH and VL, respectively) typically have similar structures, each of which contains four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al, Kuby Immunology , 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind to a particular antigen can be isolated using a VH or VL domain from an antibody that binds to the antigen to separately screen a library of complementary VL or VH domains. See, for example, Portolano et al, J. Immunol. 150: 880-887 (1993); Clarkson et al, Nature 352: 624-628 (1991).

The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are included in the genome of the host cell into which they are introduced. Certain vectors are capable of directing the performance of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression carriers."

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to certain cytotoxic cells (eg, natural killer (NK) cells, neutrophils, and giants Receptors (FcR) present on phagocytes, which enable these cytotoxic effector cells to specifically bind to target cells carrying the antigen, and subsequently kill the target cells with cytotoxin. Antibodies "arm" cytotoxic cells and require the destruction of target cells by this mechanism. Primary cells that mediate ADCC NK cells express only FcγRIII, while monocytes exhibit FcγRI, FcγRII, and FcγRIII. The performance of Fc on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu . Rev. Immunol 9:457-92 (1991). In order to assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, such as those described in U.S. Patent No. 5,500,362 or 5,821,337. Effector cells useful in such assays include peripheral blood mononuclear spheres (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo (e.g., in an animal model such as that disclosed in Clynes et al, PNAS USA 95:652-656 (1998)).

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of a typical complement pathway is initiated by binding of a first component of the complement system (Clq) to an antibody (or appropriate subclass) that binds to its cognate antigen. To assess complement activation, CDC analysis can be performed, for example as described in Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996).

The term "asthmatic" refers to a complex condition characterized by variable and relapsing symptoms, reversible airflow obstruction (eg, by bronchodilators), and bronchial hyperresponsiveness, which may or may not be associated with underlying inflammation. Examples of asthma include aspirin-sensitive/aggressive asthma, atopic asthma, severe asthma, mild asthma, moderate to severe asthma, asthma without corticosteroid treatment, chronic asthma, corticosteroid-resistant asthma, corticosteroids Refractory asthma, newly diagnosed and untreated asthma, smoking-induced asthma, uncontrolled asthma in corticosteroid use, and other asthma as mentioned in J Allergy Clin Immunol (2010) 126(5): 926-938. Symptoms of asthma include shortness of breath, cough (change in sputum production and/or sputum quality and/or cough frequency), wheezing, chest tightness, bronchoconstriction, and nighttime blame on one of the above symptoms or a combination of such symptoms Awakening (Juniper et al. (2000) Am. J. Respir. Crit. Care Med., 162(4), 1330-1334.).

The term "mild asthma" refers to a patient who typically experiences symptoms or exacerbations less than twice a week, night symptoms less than twice a month, and is asymptomatic between exacerbations. Mild intermittent asthma usually requires the following treatments: inhaled bronchodilator (short-acting inhaled β2-agonist); avoids known triggers; annual influenza vaccine; pneumococcal every 6 to 10 years, and In some cases, an inhaled beta2-agonist, cromolyn or nedocromil is administered prior to exposure to the identified trigger. If the patient has an increased need for a short-acting β2-agonist (eg, more than 3 to 4 times a short-acting β2-agonist for acute exacerbation or more than one cartridge per month for symptoms), then the patient Can need to be incremented treatment.

The term "poisoning asthma" generally refers to the following asthma: patients experience an increase in weight more than twice a week and aggravate affects sleep and activity; patients have nighttime awakening more than twice a month due to asthma; patients have a need for short-acting inhalation β2- Chronic asthma symptoms of the agonist once or every other day; and the patient's pre-treatment baseline peak expiratory flow (PEF) or 1-second maximum expiratory volume (FEV1) is predicted to be 60% to 80% and PEF variability It is 20% to 30%.

The term "severe asthma" usually refers to asthma: patients with almost persistent symptoms, frequent exacerbations, frequent nighttime awakening due to asthma, restricted activity, PEF or FEV1 baseline less than 60% predicted and PEF variability 20% to 30% .

The term "FEV1" refers to the volume of air exhaled during the first second of forced exhalation. It is a measure of airway obstruction. FEV1 may be other similar recorded, e.g. FEV _s, and the like should be understood that all such variations have the same meaning.

The term "corticosteroids" includes glucocorticoids and mineralocorticoids. For example, corticosteroids include, but are not limited to, fluticasone (including fluticasone propionate (FP)), beclometasone, budesonide, ciclesonide, momei Mometasone, flunisolide, betamethasone, hydrocortisone, prednisone, prednisolone, methylprednisolone (methylprednisolone) and triamcinolone. "Inhalable corticosteroid" means a corticosteroid that is delivered by inhalation. Exemplary inhalable corticosteroid fluticasone, beclomethasone dipropionate, budesonide, mometasone furoate, ciclesonide, flunisolide, triamcinolone acetonide, and any other cortex currently available or available in the future Steroid. Examples of corticosteroids that are inhalable and combined with long acting beta2-agonists include, but are not limited to, budesonide/formoterol and fluticasone/salmeterol.

The term " cytokine " is a generic term for a protein that is released by one cell population and acts as an intercellular medium on another cell. Examples of such cytokines are lymphokines, mononuclear factors; interleukin (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, including PROLEUKIN ^® rIL-2; tumor necrosis factor, such as TNF-α or TNF-β; and others Polypeptide factors, including LIF and kit ligands (KL). As used herein, the term cytokine includes biologically active equivalents of proteins derived from natural sources or recombinant cell cultures and natural sequence cytokines, including synthetically produced small molecule entities and pharmaceutically acceptable derivatives and salts thereof. .

The singular forms "a", "the" and "the" are used in the s For example, reference to "antibody" refers to one or more antibodies (eg, molar amounts) and includes equivalents known to those skilled in the art, and the like.

It should be understood that the aspects and embodiments of the invention described herein include "comprises" and "comprises" and "comprising".

II. Compositions and methods In one aspect, an antibody that binds to CRTh2 is provided herein. In certain embodiments, when an effective amount is administered to an individual (eg, a human individual), anti-CRTh2 binds to human CRTh2 and consumes CRTh2 expressing cells. In some embodiments, the anti-CRTh2 antibody also binds to CRTh2 (eg, rhesus or cynomolgus CRTh2) of a non-human primate. The antibodies of the invention are useful, for example, in the diagnosis or treatment of conditions mediated by CRTh2 expressing cells.

An exemplary anti-CRTh2 antibody

In one aspect, the invention provides an isolated antibody that binds to CRTh2. In certain embodiments, an anti-CRTh2 antibody has one or more of the following characteristics: (1) binding an CRTh2 (eg, human CRTh2) and consuming CRTh2 expressing cells (eg, Th2 when an effective amount is administered to an individual) Cells, mast cells, eosinophils, basophilic spheres and/or congenital type 2 (IT2) cells; (2) engineered to improve ADCC; (3) afucosylated or reduced rocks (4) competitively inhibiting the binding of at least one of the following antibodies to human CRTh2: 19A2, 8B1, 31A5, 3C12 and any of the humanized antibodies described herein; (5) binding to the following An epitope of human CRTh2 that binds to or overlaps with a CRTh2 epitope that binds to at least one of the CRTh2 antibodies: 19A2, 8B1, 31A5, 3C12, and any of the humanized antibodies described herein; (6) binds to humans and non- CRTh2 of human primates (eg, rhesus monkeys and/or cynomolgus CRTh2); (7) blocking CRTh2 signaling; (8) preventing CRTh2 expressing cells from responding to prostaglandin D2 recruitment; (9) resistance breaking performance CRTh2 cells of Ca2 ⁺ flux; (10) does not exhibit agonist activity; (11) without decreasing the CRTh2-expressing cells (e.g., performance Prostaglandin-induced cAMP content in 293 cells of CRTh2; and (12) Prostaglandin D2 triggering inhibition of forskolin-induced cAMP content in CRTh2 expressing cells (eg, 293 cells expressing human CRTh2) .

In another aspect, the invention provides an isolated anti-CRTh2 antibody comprising (a) a light chain variable region comprising at least one, two or three HVRs selected from the group consisting of murine antibody 19A2 Any of 8B1, 31A5, and 3C12 and humanized antibodies described herein (eg, hu8B1.v1, hu19A2.v1, v12, v38, v46, v47, v51-v53, v57, v58, and v60-v72) HVR-L1, HVR-L2 and HVR-L3; and/or (b) the heavy chain variable region comprises at least one, two or three HVRs selected from the group consisting of murine antibodies 19A2, 8B1, 3C12 and 31A5 HVR-H1, HVR- of any of the humanized antibodies described herein (eg, hu8B1.v1, hu19A2.v1, v12, v38, v46, v47, v51-v53, v57, v58, and v60-v72) H2 and HVR-H3. In some embodiments, HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 comprise Kabat CDR, Chothia CDR or Contact CDR sequences, as shown in Figures 10, 11A, 11B, Shown in 13 and 14.

In another aspect, the invention provides an anti-CRTh2 antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (i) HVR-L1 comprising SEQ ID NO: amino acid sequence of 22 or 23; (ii) HVR-L2, which comprises SEQ ID NO: 25 amino acid sequence; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29 or 30 (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32 or 33; (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35 or 36.

In another aspect, the invention provides an anti-CRTh2 antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (i) HVR-L1 comprising SEQ ID NO: 24 amino acid sequence; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28; Iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; (vi) HVR-H3 comprising SEQ ID NO: amino acid sequence of 37.

In another aspect, the invention provides an anti-CRTh2 antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (i) HVR-L1 comprising an amine group Acid sequence RASENIYXNLA (SEQ ID NO: 1), wherein X is S, W or Y; (ii) HVR-L2 comprising the amino acid sequence AATQLAX (SEQ ID NO: 2), wherein X is D, E or S (iii) HVR-L3 comprising the amino acid sequence QHFWITPWT (SEQ ID NO: 3); (iv) HVR-H1 comprising the amino acid sequence X ₁ YX ₂ MS (SEQ ID NO: 4), wherein X ₁ is S or F, and X ₂ is S, L or K; (v) HVR-H2 comprising an amino acid sequence X ₁ ISNGGSTTX ₂ YPGTVEG (SEQ ID NO: 5), wherein X ₁ is Y or R And X ₂ is Y or D; (vi) HVR-H3 comprising the amino acid sequence HRTNWDFDY (SEQ ID NO: 6).

In one aspect, the invention provides an antibody comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising an amino group of SEQ ID NO: 29 or 30 Acid sequence; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32 or 33; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35 or 36. In one embodiment, the antibody comprises an amino acid sequence comprising SEQ ID NO: 35 or 36 Listed in HVR-H3. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35 or 36 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. In still another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35 or 36, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and comprising SEQ ID NO: 32 or HVR-H2 of the amino acid sequence of 33. In still another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29 or 30; (b) HVR-H2 comprising the amino group of SEQ ID NO: 32 or 33 An acid sequence; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35 or 36.

In another aspect, the invention provides an antibody comprising at least one, at least two or all three VL HVRs selected from the group consisting of: (a) HVR-L1 comprising an amino group of SEQ ID NO: 22 or 23. Acid sequence; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22 or 23; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; And (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:27.

In one aspect, the invention provides an antibody comprising at least one, at least two or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31 (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:37. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:37. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28. In still another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28, and an amine group comprising SEQ ID NO: Acid sequence HVR-H2. In still another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34; And (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:37.

In another aspect, the invention provides an antibody comprising at least one, at least two or all three VL HVRs selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24. (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:28.

In one aspect, the invention provides an antibody comprising at least one, at least two or all three VH HVR sequences selected from: (a) HVR-H1 comprising SEQ ID NOs: 13, 14, 15, a 16 or 17 amino acid sequence; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, 19, 20 or 21; and (c) HVR-H3 comprising SEQ ID NO: 6 Amino acid sequence. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the antibody comprises HVR-H3 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, which comprises the amino acid sequence of SEQ ID NO: 3. In a further embodiment, the antibody comprises HVR-H3, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6, comprising the amino acid sequence of SEQ ID NO: 3 and comprising SEQ ID NO: 18, 19 HVR-H2 of the amino acid sequence of 20 or 21. In still another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16 or 17; (b) HVR-H2 comprising SEQ ID NO: An amino acid sequence of 18, 19, 20 or 21; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6.

In another aspect, the invention provides an antibody comprising at least one, at least two or all three VL HVRs selected from: (a) HVR-L1 comprising SEQ ID NO: 7, 8, or 9. An amino acid sequence; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, 11 or 12; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, the antibody comprises (a) HVR-L1 comprising SEQ ID NOs: 7, 8 Or an amino acid sequence of 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, 11 or 12; and (c) HVR-L3 comprising the amino group of SEQ ID NO: Acid sequence.

In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising SEQ ID NO: a 29 or 30 amino acid sequence, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32 or 33, and (iii) HVR-H3 comprising a selected from the group consisting of SEQ ID NO: 35 or 36 And the amino acid sequence comprising: at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising SEQ ID NO: 22 or 23. An amino acid sequence, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25 and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another aspect, the invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29 or 30; (b) HVR-H2 comprising SEQ ID NO: An amino acid sequence of 32 or 33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35 or 36; (d) HVR-L1 comprising the amino group of SEQ ID NO: 22 or 23. Acid sequence; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising SEQ ID NO: An amino acid sequence of 31, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 and (iii) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 37; (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (ii) HVR -L2 comprising the amino acid sequence of SEQ ID NO: 26 and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

In another aspect, the invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising SEQ ID NO: 34 amino acid sequence; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

In another aspect, the antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from: (i) HVR-H1 comprising SEQ ID NO An amino acid sequence of 13, 14, 15, 16 or 17, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, 19, 20 or 21 and (iii) HVR-H3, Included is an amino acid sequence selected from the group consisting of SEQ ID NO: 6; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from: (i) HVR-L1, comprising SEQ ID NO: amino acid sequence of 7, 8 or 9, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, 11 or 12 and (c) HVR-L3 comprising SEQ ID NO: amino acid sequence of 3.

In another aspect, the invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16 or 17; (b) HVR-H2, It comprises the amino acid sequence of SEQ ID NO: 18, 19, 20 or 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (d) HVR-L1 comprising SEQ ID NO: amino acid sequence of 7, 8 or 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NOS: 10, 11, 12; and (f) HVR-L3 comprising SEQ ID NO: amino acid sequence of 3. In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (e) HVR-L2 comprising SEQ ID NO An amino acid sequence of 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: Column (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (e) HVR-L2, An amino acid sequence comprising SEQ ID NO: 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising SEQ ID NO An amino acid sequence of 11; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2 comprising SEQ ID NO An amino acid sequence of 10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3.

In any of the above embodiments, the anti-CRTh2 anti-system is isolated from the antibody. In any of the above embodiments, the anti-CRTh2 antibody is humanized. In one embodiment, the anti-CRTh2 antibody comprises an HVR as in any of the above embodiments and an HVR as shown in Figures 10, 11, 12, 13 and 14 (including comprising a Kabat CDR, Chothia CDR or Contact CDR sequence) HVR), and further comprises a receptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-CRTh2 antibody comprises an HVR as in any of the above embodiments, and further comprising an FR (eg, FR1, FR2, FR3, or FR4) as shown in Figures 11A, 12, and 14A. The VL of the sequence. In another embodiment, the anti-CRTh2 antibody comprises an HVR as in any of the above embodiments and an HVR as shown in Figures 10, 11, 12, 13 and 14 (including comprising a Kabat CDR, Chothia CDR or Contact CDR sequence HVR), and further comprising a VH comprising a sequence of FR (eg, FR1, FR2, FR3 or FR) as shown in Figures 11B, 12 and 14B.

In certain embodiments, an anti-CRTh2 antibody described herein comprises an HVR as defined by Kabat, eg, an anti-CRTh2 antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 , each of which is defined by Kabat, as further described herein. In certain embodiments, an anti-CRTh2 antibody described herein comprises an HVR as defined by Chothia, eg, an anti-CRTh2 antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 , each of which is defined by Chothia, as further described herein. In certain embodiments, an anti-CRTh2 antibody described herein comprises an HVR as defined by a Contact CDR sequence, eg, an anti-CRTh2 antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR -L3, wherein each CDR is defined by a Contact CDR sequence, as further described herein.

In another aspect, an anti-CRTh2 antibody is provided, wherein the antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96 with the amino acid sequence of SEQ ID NOs: 38-53 %, 97%, 98%, 99% or 100% sequence-consistent light chain variable domain (VL). In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence contains a substitution ( For example, a conservative substitution), insertion or deletion, but an anti-CRTh2 antibody comprising the sequence retains the ability to bind to CRTh2. In certain embodiments, in any one of SEQ ID NOs: 38-53, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have been substituted, inserted, and / or missing. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (ie, in the FR). Optionally, the anti-CRTh2 antibody comprises a VL sequence selected from the group consisting of SEQ ID NOs: 38-53, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from the group consisting of: (i) HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 7-9; HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 10-12; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In a specific embodiment, the VL comprises one, two or three HVRs selected from the group consisting of: (i) HVR-L1 comprising the SEQ ID NO: 22 or 23 amino acid sequence; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (iii) HVR-L3 comprising the amino acid of SEQ ID NO: 27. sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (ii) HVR-L2, comprising The amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

In another aspect, the anti-CRTh2 antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and the amino acid sequence of SEQ ID NOs: 54-65, 98%, 99% or 100% sequence identity heavy chain variable domain (VH) sequence. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence contains a substitution ( For example, a conservative substitution), insertion or deletion, but the anti-CRTh2 antibody comprising the sequence retains the ability to bind to CRTh2. In certain embodiments, in any of SEQ ID NOs 54-65, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are substituted, inserted, and / or missing. In certain embodiments, substitutions, insertions, or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-CRTh2 antibody comprises the VH sequence of SEQ ID NOs: 54-65, including post-translational modifications of the sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 13-17, (b) HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 18-21, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In a particular embodiment, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29 or 30, (b) HVR-H2, It comprises the amino acid sequence of SEQ ID NO: 32 or 33, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35 or 36. In a particular embodiment, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31, (b) HVR-H2, comprising Amino acid sequence of SEQ ID NO: 34 Column, and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:37.

In another aspect, an anti-CRTh2 antibody is provided, wherein the antibody comprises a VH of any of the embodiments provided above and a VL of any of the embodiments provided above. In some embodiments, the antibody comprises any one of murine antibodies 8B1, 3C12, 31A5, and 19A2 and humanized antibody hu19A2 (including v1, v12, v38, v46, v47, v51-v53, v57, v58, and v60) -v72) VH sequence. In some embodiments, the antibody comprises any one of murine antibodies 8B1, 3C12, 31A5, and 19A2 and humanized antibody hu19A2 (including v1, v12, v38, v46, v47, v51-v53, v57, v58, and v60-) V72) VL sequence. In one embodiment, the antibody comprises a VH sequence selected from the group consisting of SEQ ID NOs: 54-60 and a VL sequence selected from the group consisting of SEQ ID NOs: 38-48, including post-translational modifications of such sequences. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 55 and the VL sequence of SEQ ID NO: 39, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 57 and the VL sequence of SEQ ID NO: 41, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 61 and the VL sequence of SEQ ID NO: 49, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 50, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 63 and the VL sequence of SEQ ID NO: 51, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 64 and the VL sequence of SEQ ID NO: 52, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 65 and the VL sequence of SEQ ID NO: 53, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 57 and the VL sequence of SEQ ID NO: 40, including post-translational modifications of the sequences.

In yet another aspect, the invention provides an antibody that binds to the same epitope as an anti-CRTh2 antibody provided herein. For example, in certain embodiments, providing antibodies to murine animals 8B1, 3C12, 31A5 and 19A2 and the humanized antibody hu19A2 (including v1, v12, v38, v46, v47, v51-v53, v57, v58 and v60-v72) bind to antibodies of the same epitope.

In yet another aspect, an anti-CRTh2 antibody that binds to both human CRTh2 and at least one non-human primate CRTh2 is provided. In certain embodiments, the anti-CRTh2 antibody binds to human CRTh2 and cynomolgus CRTh2. In certain embodiments, the anti-CRTh2 antibody binds to human CRTh2 and rhesus CRTh2. In certain embodiments, the anti-CRTh2 antibody binds to human CRTh2, rhesus monkey CRTh2, and cynomolgus CRTh2. In certain embodiments, the anti-CRTh2 antibody K _D of less than 100nM binding to both human CRTh2 and at least one non-human primates CRTh2 (e.g., an anti-CRTh2 of less than 100nM antibody bound to the K _D and with human CRTh2 K _{D of} less than 100 nM binds to at least one non-human primate CRTh2). In certain embodiments, the anti-antibody CRTh2 of less than 75nM, 50nM, 45nM, 40nM, 35nM, 30nM, 25nM, 20nM, 15nM or 10nM K _D of binding to a human CRTh2 both and at least one non-human primates CRTh2 . In certain embodiments, an anti-CRTh2 anti-systemic depleting antibody that binds to both human CRTh2 and at least one non-human primate CRTh2, eg, an antibody that depletes CRTh2 expressing cells is further set forth herein.

In another aspect of the invention, the anti-CRTh2 anti-system monoclonal antibody of any of the above embodiments comprises a chimeric, humanized or human antibody. In one embodiment, an anti-CRTh2 anti-system antibody fragment, such as an Fv, Fab, Fab', scFv, diabody or F(ab') ₂ fragment. Embodiment, the antibody is a full length antibody In another embodiment, for example, or a complete IgG1 antibody as defined herein, other classes of antibodies or isotype (e.g., IgG _2, IgG ₃ or IgG _4). In some embodiments, the antibody comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 77-83; and/or a light chain sequence selected from the group consisting of SEQ ID NOs: 66-76.

In still another aspect, the anti-CRTh2 antibody of any of the above embodiments may be included, either alone or in combination, in any of the features described in the following section:

Antibody affinity

In certain embodiments, the dissociation constant (Kd) of an antibody provided herein 1μM, 150nM, 100nM, 50nM, 10nM, 1nM, 0.1nM, 0.01nM or 0.001 nM (eg 10 ^-8 M or less, such as 10 ^-8 M to 10 ^-13 M, such as 10 ^-9 M to 10 ^-13 M).

In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA) using Fab forms of the antibody of interest and antigens thereof as described in the following assays. The solution binding affinity of the Fab to the antigen is measured by equilibrating the Fab to a minimum concentration of the ( ^125I ) labeled antigen in the presence of a titrated series of unlabeled antigens, and then coating it with an anti-Fab antibody. The plate captures the bound antigen (see, eg, Chen et al, J. Mol. Biol. 293:865-881 (1999)). Conditions for the establishment of the perforated plate MICROTITER ^® (Thermo Scientific) with 5μg / ml in 50mM sodium carbonate stored (pH 9.6) in the capturing anti-Fab antibody (Cappel Labs) coated overnight at room temperature and then ( Blocked with 2% (w/v) bovine serum albumin in PBS for 2 hours to 5 hours at approximately 23 °C. In a non-absorbable plate (Nunc #269620), 100 pM or 26 pM [ ¹²⁵ I] antigen is mixed with serial dilutions of the Fab of interest (for example, against Presta et al, Cancer Res. 57: 4593-4599 (1997) The evaluation of the VEGF antibody Fab-12 was consistent). The Fab of interest is then incubated overnight; however, incubation can be continued for a longer period of time (e.g., about 65 hours) to ensure equilibrium is achieved. The mixture is then transferred to a capture plate for incubation at room temperature (eg, 1 hour). Solution is then removed and used in PBS with 0.1% of polysorbate 20 (TWEEN-20 ^®) The plates were washed 8 times. When the plate has been dried, added 150μl / scintillator (MICROSCINT-20 ^TM; Packard) of the hole, and the other plate ^(TM) counts of gamma] counter on a TOPCOUNT (Packard) 10 min. The concentration of each Fab that achieved a maximum binding of less than or equal to 20% was selected for competitive binding analysis. In some embodiments, Kd can also be measured for binding of the antibody to CRTh2 expressed on the cell surface.

According to another embodiment, the use of surface plasmon resonance analysis using BIACORE ^® -2000 or a BIACORE ^® -3000 (BIAcore Company, Piscataway, NJ) with immobilized antigen CM5 chip at ~ 10 response units (RU) of at 25 deg.] C To measure Kd. Briefly, N -ethyl- N' -(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N -hydroxysuccinimide (NHS) were used according to the supplier's instructions. To activate a carboxymethylated dextran biosensor wafer (CM5, BIACORE). The antigen was diluted to 5 μg/ml (about 0.2 μM) using 10 mM sodium acetate (pH 4.8), and then injected at a flow rate of 5 μl/min to achieve about 10 reaction units (RU) of coupled protein. After antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, at about 25 deg.] C at a flow rate of 25μl / min stored in the injection containing 0.05% Polysorbate 20 (TWEEN-20 ^TM) PBS surfactant (PBST) of the two-fold serial dilutions of Fab (0.78nM to 500nM). Association rate (k _on) and dissociation rates (k _off) system using a simple one Langmuir binding model (BIACORE ^® Evaluation Software version 3.2) by simultaneous fitting the association and the solution is calculated from the sensed FIG. The equilibrium dissociation constant (Kd) is calculated as the ratio k _off /k _on . See, for example, Chen et al, J. Mol. Biol. 293:865-881 (1999). If the association rate measured by the above surface plasmon resonance analysis exceeds 10 ⁶ M ^-1 S ^-1 , the association rate can be determined by using a fluorescence quenching technique, which is increased at 25 ° C. The increase or decrease in the fluorescence emission intensity of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) in the presence of a concentration of antigen (excitation = 295 nm; emission = 340 nm, 16 nm band pass), as in with the interrupter, such as a spectrophotometer (Aviv Instruments) or with a stirred cuvette amount of 8000- series SLM-AMINCO ^TM spectrophotometer (ThermoSpectronic) measured in the spectrophotometer.

Antibody fragment

In certain embodiments, anti-system antibody fragments are provided herein. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv and scFv fragments and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies , Vol. 113, Rosenburg and Moore ed. (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments comprising a salvage receptor binding epitope residue and having an increased in vivo half-life, see U.S. Patent No. 5,869,046.

A bispecific anti-system has two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9: 129-134 (2003); and Hollinger et al, Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) . Tri-chain antibodies and four-chain antibodies are also described in Hudson et al, Nat. Med. 9: 129-134 (2003).

A single domain anti-system comprises antibody fragments that comprise all or a portion of a heavy chain variable domain of an antibody or all or a portion of a light chain variable domain. In certain embodiments, a single domain is directed against a systemic human single domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1).

Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

Chimeric and humanized antibodies

In certain embodiments, an anti-system chimeric antibody is provided herein. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA , 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (eg, a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (eg, a monkey)) and a human constant region. In yet another example, a chimeric anti-system class or subclass has a "class-switching" antibody that has been altered from a parent antibody. A chimeric antibody comprises an antigen binding fragment thereof.

In certain embodiments, the chimeric anti-system humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the parental non-human antibodies. Typically, humanized antibodies contain one or more variable domains, of which HVR (eg, CDR) (or a portion thereof) is derived from a non-human antibody, and FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody may optionally comprise at least a portion of a human constant region. In some embodiments, some of the FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (eg, an antibody that produces an HVR residue) to, for example, restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and further described, for example, in Riechmann et al, Nature 332:323- 329 (1988); Queen et al . Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (Explaining SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (explaining "surface remodeling");Dall'Acqua et al., Methods 36:43-60 (2005) (Explaining "FR Reorganization"); and Osbourn et al, Methods 36: 61-68 (2005) and Klimka et al, Br . J. Cancer , 83: 252-260 (2000) ( Explain the "guided selection" method for FR reorganization).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, for example, Sims et al. , J. Immunol. 151:2296 (1993)); Or a framework region of a consensus sequence of human antibodies of a particular subgroup of heavy chain variable regions (see, eg, Carter et al, Proc. Natl. Acad. Sci. USA , 89: 4285 (1992); and Presta et al, J. Immunol. , 151: 2623 (1993)); human maturation ( transformation ) framework regions or human germline framework regions (see, eg, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and self-screening The framework region obtained by the FR library (see, for example, Baca et al, J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al, J. Biol. Chem. 271: 22611-22618 (1996)).

Human antibody

In certain embodiments, anti-system human antibodies are provided herein. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol . 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol . 20: 450-459 (2008).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce an intact human antibody or an intact antibody having a human variable region that is responsive to antigenic challenge. Such animals typically contain all or a portion of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosome. In these transgenic mice, the endogenous immunoglobulin loci are generally not activated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). For example, see also U.S. Pat. No. 6,075,181 and No. 6,150,584, which describes XENOMOUSE ^TM technologies; U.S. Patent No. 5,770,429, which describes techniques HUMAB®; U.S. Patent No. 7,041,870, which describes techniques KM MOUSE®; and U.S. Patent Application Publication No. US 2007/0061900, which describes VELOCIMOUSE® technology. Human variable regions of intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

Human antibodies can also be prepared by fusion-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol. , 133: 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J .Immunol. , 147:86 (1991). Human antibodies produced by human B cell fusion tumor technology are also described in Li et al, Proc. Natl. Acad. Sci. USA , 103: 3557-3562 (2006). Additional methods include those described in, for example, U.S. Patent No. 7,189,826 (which describes the production of a single human IgM antibody from a fusion tumor cell line) and Ni, Xiandai Mianyixue , 26(4): 265-268 (2006) - Human fusion tumor). Human fusion tumor technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology , 20(3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology , 27 (3): 185-91 (2005).

Human antibodies can also be produced by isolating Fv pure line variable domain sequences selected from human phage display libraries. These variable domain sequences can then be combined with the desired human constant domain. Techniques for selecting human antibodies from antibody libraries are set forth below.

Antibody derived from the library

An antibody of the invention can be isolated by screening a combinatorial library for antibodies having one or more desired activities. For example, various methods are known in the art for producing phage display libraries and screening antibodies having the desired binding properties from such libraries. Such methods are discussed, for example, in Hoogenboom et al, Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further illustrated in, for example, the following: McCafferty et al, Nature 348: 552-554; Clackson et al, Nature 352: 624-628 (1991); Marks et al, J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al, J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol .Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284 ( 1-2): 119-132 (2004).

In some phage display methods, the VH and VL spectra are separately selected by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then antigen-binding phage can be screened, such as Winter et al., Ann . Rev .Immunol. , 12: 433-455 (1994). Phage typically display antibody fragments that are single-chain Fv (scFv) fragments or are Fab fragments. A library from an immunogenic source can provide high affinity antibodies to the immunogen without the need to construct a fusion tumor. Alternatively, the natural spectrum (e.g., from humans) is selected to provide a single source of antibodies to various non-autoantigens and autoantigens without any immunization, as by Griffiths et al, EMBO J, 12: 725-734 (1993). Said. Finally, natural libraries can also be made synthetically by: culturing unrearranged V-gene fragments from stem cells, and encoding highly variable CDR3 regions using PCR primers containing random sequences and rearranging in vitro, As described by Hoogenboom and Winter, J. Mol. Biol. , 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

An antibody or antibody fragment isolated from a human antibody library can be considered a human antibody or a human antibody fragment herein.

Multispecific antibody

In certain embodiments, anti-system multispecific antibodies, such as bispecific antibodies, are provided herein. A multispecific antibody that has binding specificity for at least two different sites. In certain embodiments, one of the binding specificities is for CRTh2 and the other is for any other antigen. In certain embodiments, a bispecific antibody can bind to two different epitopes of CRTh2. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express CRTh2. Bispecific antibodies can be made in the form of full length antibodies or antibody fragments.

Techniques for preparing multispecific antibodies include, but are not limited to, recombinantly displaying two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/ 08829 and Traunecker et al., EMBO J. 10:3655 (1991)) and "knob-in-hole" modification (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by modifying the electrostatic steering effect for the preparation of antibody Fc-heterodimer molecules (WO 2009/089004 A1); making two or more antibodies or Fragment cross-linking (see, for example, U.S. Patent No. 4,676,980, and Brennan et al, Science , 229:81 (1985)); leucine zippers are used to generate bispecific antibodies (see, for example, Kostelny et al. , J. Immunol. , 148(5): 1547-1553 (1992)); the use of "double-stranded antibody" technology for the preparation of bispecific antibody fragments (see, eg, Hollinger et al, Proc. Natl. Acad. Sci. USA , 90:6444- 6448 (1993)); and using single-chain Fv (sFv) dimers (for example, see Gruber et al, J. Immunol. 152: 5368 (1994)); and preparing trispecific antibodies such as, for example, Tutt et al. Human, J. Immunol. 147: 60 (1991).

Also included herein are engineered antibodies (including "octopus antibodies") having three or more functional antigen binding sites (see, for example, US 2006/0025576 A1).

An antibody or fragment herein also includes a "dual-acting FAb" or "DAF" comprising an antigen binding site that binds to CRTh2 and another different antigen (see, for example, US 2008/0069820).

Antibody variant

In certain embodiments, the invention encompasses amino acid sequence variants of the antibodies provided herein. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be implemented to achieve the final construct, provided that the final construct has desirable properties, such as antigen binding.

Substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include HVR and FR. Conservative substitution lines are shown under the heading "Conservative substitutions" in Table 1. Other substantial changes are shown under the heading "Example Substitutions" in Table 1, and are further described below with reference to the amino acid side chain classes. Amino acid substitution can be introduced into the antibody of interest and the product for screening for the desired activity, the desired activity Sex lines, for example, maintain/improve antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Amino acids can be grouped according to common side chain properties: a. Hydrophobicity: n-leucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; c. Acidity: Asp, Glu; d. Basic: His, Lys, Arg; e. Residues affecting chain orientation: Gly, Pro; f. Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will necessitate the exchange of members of one of these categories with another.

One type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Generally, the resulting variants selected for further study have modifications (eg, improvements) and/or substantial pro-relationships in certain biological properties (eg, increased affinity, reduced immunogenicity) relative to the parent antibody. Some biological properties of the generation of antibodies. Exemplary substituted system affinity matured antibodies, which can be conveniently produced, for example, using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for specific biological activities (eg, binding affinity).

Alterations (e.g., substitutions) can be made in the HVR to, for example, improve antibody affinity. Such changes may be at the HVR "hot spot" (i.e., residues encoded by codons that undergo mutations at high frequencies during the somatic maturation process) (see, for example, Chowdhury, Methods Mol. Biol. 207: 179-196 (2008). () and / or SDR (a-CDR) were carried out in which the binding affinities of the resulting variants VH or VL were tested. Affinity maturation by constructing and reselecting from secondary libraries has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., edited by Human Press, Totowa, NJ, (2001). ))in. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (eg, error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis) in. A secondary library is then produced. The library is then screened to identify any antibody variant with the desired affinity. Another method of introducing diversity involves an HVR-directed approach in which several HVR residues (eg, 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are typically targeted.

In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs as long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions provided herein) can be made in the HVR that do not substantially reduce binding affinity. These changes can be located outside of the HVR "hotspot" or SDR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered or contains no more than one, two or three amino acid substitutions.

A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science , 244: 1081-1085. In this method, a population of residues or target residues (eg, charged residues such as arg, asp, his, lys, and glu) is identified and neutralized by a neutral or negatively charged amino acid (eg, alanine or Instead of measuring whether the antibody interacts with the antigen, it is replaced by polyalanine. Other substitutions can be introduced at the position of the amino acid that demonstrates sensitivity to the initial substitution function. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the point of contact between the antibody and the antigen. The contact residues and adjacent residues can be targeted as replacement candidates or excluded. Variants can be screened to determine if they contain the desired properties.

Amino acid sequence insertions include intra- and/or carboxy-terminal fusions in the range of one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having N-terminal methionine residues. Other insertional variants of the antibody molecule include a polypeptide in which the N-terminus or C-terminus of the antibody is fused to an enzyme (eg, for ADEPT) or which increases the serum half-life of the antibody.

Glycosylation variant

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. One or more glycosylation sites can be created or removed by altering the amino acid sequence It is convenient to achieve the addition or deletion of a glycosylation site to an antibody.

If the antibody comprises an Fc region, the carbohydrate to which it is attached may vary. Native antibodies produced by mammalian cells typically comprise a branched, branched oligosaccharide that is typically attached to the Asn297 of the CH2 domain of the Fc region by an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates such as mannose, N-ethyl glucosamine (GlcNAc), galactose and sialic acid, and fucose of GlcNAc attached to the "backbone" of the double-antenna oligosaccharide structure. . In some embodiments, an oligosaccharide in an antibody of the invention can be modified to produce an antibody variant having certain improved properties.

In one embodiment, an antibody variant comprising an Fc region is provided, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function. Specifically, the present invention encompasses antibodies having reduced fucose relative to the amount of fucose on the same antibody produced in wild-type CHO cells. In other words, it is characterized by having a fucose amount which is lower than the amount of fucose originally to be produced by natural CHO cells (for example, CHO cells which produce a natural glycosylation pattern, such as CHO cells containing the native FUT8 gene). In certain embodiments, the anti-system comprises less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans comprising fucose. For example, the amount of fucose in the antibody may range from 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. In certain embodiments, the anti-system does not contain fucose on the N-linked glycan, ie, wherein the antibody is completely free of fucose, or has no fucose or is afucosylated. The amount of fucose is determined by calculating the average amount of fucose at Asn297 in the sugar chain relative to the sum of all sugar structures attached to Asn 297 (eg, complex, heterozygous, and high mannose structures), such as Measured by MALDI-TOF mass spectrometry as described, for example, in WO 2008/077546. Asn297 refers to the aspartame residue located at about 297 positions in the Fc region (Eu number of the Fc region residue); however, Asn297 may also be located upstream or downstream of the 297 position due to a slight sequence change in the antibody. The three amino acids are between 294 and 300. Such fucosylated variants can have improved ADCC function. See, for example, U.S. Patent Publication No. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of disclosures relating to "defucosylation" or "lack of fucose" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328 US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO 2002/031140; Okazaki et al, J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al, Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al, Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al, especially in Example 11) and gene knockout cell lines, such as the α-1,6-fucosyltransferase gene FUT8 knocks out CHO cells (see, for example, Yamane-Ohnuki et al, Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al . Biotechnol. Bioeng. , 94(4): 680-688 (2006); WO2003/085107).

Further provided are antibody variants having a bisecting oligosaccharide, for example, wherein the dual antenna oligosaccharide attached to the Fc region of the antibody is halved by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); US 2005/0123546 (Umana et al.) and Ferrara et al., Biotechnology. And Bioengineering, 93(5): 851-861 (2006). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); 1999/22764 (Raju, S.).

In certain embodiments, an antibody variant comprising an Fc region described herein is capable of binding to FcyRIII. In certain embodiments, an antibody variant comprising an Fc region described herein has ADCC activity in the presence of a human effector cell or is comparable to an antibody comprising a human wild-type IgGl Fc region in human effector cells. There is an increased ADCC activity in the presence.

Fc region variant

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. The Fc region variant may comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (eg, a substitution) at one or more amino acid positions.

In certain embodiments, the invention encompasses antibody variants having some, but not all, effector functions, which makes them desirable candidates for many applications in which the in vivo half-life of the antibody is important, but Certain effector functions (such as complement and ADCC) systems are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/consumption of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcyR binding ability (and thus may lack ADCC activity), but retains FcRn binding ability. Primary cells of NK cells used to mediate ADCC express only FcγRIII, whereas mononuclear spheres express FcγRI, FcγRII, and FcγRIII. The performance of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu . Rev. Immunol 9:457-492 (1991). Non-limiting examples of in vitro analysis to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (for example, see Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 ( 1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); 5, 821, 337 (see Bruggemann, M. et al. , J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, a non-radioactive analysis methods (e.g., flow cytometry, the ACTI ^TM see non-radioactive cytotoxicity assay for (CellTechnology Corporation, Mountain View, CA); and CytoTox 96 ^® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Effector cells that can be used in such assays include peripheral blood mononuclear spheres (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al, Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind to C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC analysis can be performed (see, for example, Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996); Cragg, MS et al, Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance/half life assays can also be performed using methods known in the art (see, for example, Petkova, SB et al, Int'l. Immunol. 18(12): 1759-1769 (2006)).

Antibodies having reduced effector functions include those having one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of the amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" in which residues 265 and 297 are substituted with alanine. Fc mutant (U.S. Patent No. 7,332,581).

Certain antibody variants with improved or reduced binding to FcR are set forth. (See, for example, U.S. Patent No. 6,737,056, WO 2004/056312, and Shields et al, J. Biol. Chem. 9(2): 6591-6604 (2001)).

In certain embodiments, an antibody variant comprises an Fc region having one or more amino acid substitutions that modify ADCC, eg, a substitution at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues) ). In an exemplary embodiment, the anti-CRTh2 antibody comprises the following amino acid substitutions in its Fc region: S298A, E333A, and K334A.

In some embodiments, the Fc region is altered to alter (ie, improve or reduce) C1q binding and/or complement dependent cytotoxicity (CDC), for example, as in US Patent No. 6,194,551, WO 99/51642 And as described in Idusogie et al. , J. Immunol. 164: 4178-4184 (2000).

Has an increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J The antibodies of Immunol. 24: 249 (1994) are described in US 2005/0014934 A1 (Hinton et al.). These antibodies comprise a substituted Fc region having one or more modified Fc regions in combination with FcRn. The Fc variants include those having one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, replaces the Fc region residue 434 (U.S. Patent No. 7,371,826).

See also, Duncan and Winter, Nature 322: 738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351, for other examples of Fc region variants.

Cysteine-modified antibody variant

In certain embodiments, it may be desirable to produce a cysteine engineered antibody, such as a "thiocarba", wherein one or more residues of the antibody are substituted with a cysteine residue. In a particular embodiment, the substituted residue occurs at an accessible site based on the antibody. By substituting their residues with cysteine, the reactive thiol group is thus located at the accessible site of the antibody and can be used to couple the antibody to other moieties (eg, drug moiety or linker-drug moiety) To generate an immunoconjugate, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 of the heavy chain Fc region ( EU number). The cysteine-modified antibody can be produced as described in, for example, U.S. Patent No. 7,521,541.

Antibody derivative

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatizing antibodies include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrole Pyridone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) And dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polyoxypropylene/ethylene oxide copolymer, polyoxyethylated polyol (for example, glycerin), Polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde is advantageous in terms of manufacturing because of its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The amount of polymer attached to the antibody can vary, and if more than one polymer is attached, it can be the same or different molecules. In general, the amount and/or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be modified, and whether the antibody derivative will be used under defined conditions. For therapy, etc.

In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can have any wavelength and includes, but is not limited to, radiation of a wavelength that does not harm normal cells, but heats the non-proteinaceous portion to a temperature at which cells adjacent to the antibody-non-proteinaceous portion can be killed.

Recombination method and composition

Recombinant methods and compositions can be used to produce antibodies, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-CRTh2 antibody described herein is provided. The nucleic acid can encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising a VH (eg, a light chain and/or a heavy chain of an antibody). In yet another embodiment, one or more vectors (eg, expression vectors) comprising the nucleic acid are provided. In yet another implementation In the case, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (eg, has been transformed with such a substance): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amine comprising the VH of the antibody a base acid sequence, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (eg, Y0, NSO, Sp20 cells). In one embodiment, a method of making an anti-CRTh2 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as set forth above, and optionally from a host cell (or host cell) under conditions suitable for expression of the antibody Medium) recovers antibodies.

For recombinant production of an anti-CRTh2 antibody, the nucleic acid encoding the antibody, as described above, is isolated and inserted into one or more vectors for further selection and/or expression in a host cell. This nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

Suitable host cells for the selection or expression of vectors encoding the antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For the performance of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology , Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the performance of antibody fragments in E. coli). After performance, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also used in the appropriate selection or expression host of antibody-encoding vectors, including glycosylation pathways that have been "humanized" to produce partially or fully human sugars. Fungal and yeast strains of antibodies in the basic mode. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al, Nat. Biotech. 24: 210-215 (2006).

Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains that can be used in combination with insect cells have been identified, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. For example, see U.S. Pat. Nos. 5,959,177, No. 6,040,498, No. 6,420,548, No. 7,125,978 and No. 6,417,429 (describes antibodies generated PLANIBODIES ^TM technology in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are the monkey kidney CV1 cell line transformed with SV40 (COS-7); human embryonic kidney cell line (293 or 293 cells such as, for example, Graham et al., J. Gen Virol. 36) . :59 (1977); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, eg, for example, Mather, Biol. Reprod. 23:243-251 (1980) ()); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); large kidney cells (MDCK); Buffalo rat liver cells (buffalo rat liver Cell) (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells such as, for example, Mather et al., Annals NYAcad. Sci. 383:44 -68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines, for example Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology , Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). .

analysis

The anti-CRTh2 antibodies provided herein can be identified, screened, or characterized for physical/chemical properties and/or biological activity by various assays known in the art.

Combined analysis and other analysis

In one aspect, for example, the antigen-binding activity of the antibody of the present invention is tested by a known method (e.g., ELISA, Western blot, etc.).

In another aspect, competition assays can be used to identify antibodies to murine antibodies 8B1, 3C12, 31A5, and 19A2 and humanized antibodies hu19A2 (including v1, v12, v38, v46, V47, v51-v53, v57, v58 and V60-v72) an antibody that competes for binding to CRTh2. In certain embodiments, the competing antibody binds to murine antibodies 8B1, 3C12, 31A5, and 19A2 and the humanized antibody hu19A2 (including v1, v12, v38, v46, v47, v51-v53, v57, v58, and v60) -v72) the same epitope (eg, linear or conformational epitope) to which it is bound. A detailed exemplary method for mapping epitopes to which antibodies are bound is provided in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, cells that immobilize CRTh2 or express CRTh2 on the cell surface are included in a first labeled antibody (eg, a human or non-human primate) that binds to CRTh2 and are tested for competition with the first antibody. Incubation in a solution of a second unlabeled antibody that binds to the ability of CRTh2. The second antibody can be present in the supernatant of the fusion tumor. As a control, cells immobilized with CRTh2 or expressing CRTh2 were cultured in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions allowing the binding of the first antibody to CRTh2, excess unbound antibody is removed and the amount of label associated with immobilized CRTh2 or cells expressing CRTh2 is measured. If the amount of the label associated with the immobilized CRTh2 or the cell expressing CRTh2 is substantially reduced in the test sample relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to CRTh2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Activity analysis

An assay known in the art and described herein (e.g., Example 1) can be used to identify and test the biological activity of an anti-CRTh2 antibody. In some embodiments, an assay for testing an anti-CRTh2 antibody that depletes CRTh2 expressing cells (eg, Th2 cells, mast cells, eosinophils, basophilic spheres, and/or congenital type 2 (IT2) cells) is provided. Exemplary tests for biological activity can include, for example, transgenic mice that provide human CRTh2 on immune cells (eg, basophilic and eosinophilic), administer anti-CRTh2 antibodies to transgenic mice, and measure mice. The amount (eg, amount or percentage) of human CRTh2 positive cells in blood or tissue or the amount (eg, number or percentage) of cell types known to express CRTh2 in the blood or tissue of a mouse. Another exemplary test can include, for example, providing a mouse that exhibits human CRTh2, sensitizing/challenging the mouse using TNP-OVA using known methods, followed by administration of an anti-CRTh2 antibody. TNP-OVA challenged mouse lung tissue, blood, BAL and BALF can be assessed for the presence of CRTh2 positive cells or for the presence of cell types known to express CRTh2. In some embodiments, an assay for detecting consumption of a Th2 cytokine producing cell by an anti-CRTh2 antibody is provided. For example, in vitro polarized human Th2 cells can be injected intraperitoneally into SCID mice and anti-CRTh2 antibodies can be administered to such mice. The amount of cytokine-producing cells was analyzed after ex vivo stimulation with PMA and ionomycin. In some embodiments, in any of the assays, the anti-CRTh2 antibody can consume at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100% CRTh2 in the cells. Either.

An anti-test CRTh2 antibody was also provided for blocking the analysis of CRTh2 signaling. An exemplary method for assessing CRTh2 signaling can include providing CRTh2 positive cells, culturing the cells with an anti-CRTh2 antibody, followed by stimulation with a ligand such as PGD2 (in the presence or absence of forskolin), and finally by Any method known in the art measures changes in intracellular cAMP or Ca ²⁺ content.

Anti-CRTh2 antibodies are also provided to prevent the analysis of CRTh2 expressing cells in response to recruitment of TNP-OVA, papain or prostaglandin D2. An exemplary test for CRTh2 expressing cells in response to recruitment of PGD2 can include administering PGD2 to transgenic mice expressing human CRTh2 on immune cells (eg, basophilic and eosinophils) in the presence or absence of an anti-CRTh2 antibody. The subsequent influx of CRTh2 positive cells into lung tissue and bronchoalveolar lavage fluid was assessed in the airways. Evaluation can be accomplished in a number of ways, including staining the resected tissue of CRTh2 and determining cell influx via flow cytometry or any other method known in the art.

Analysis of the inhibition of Ca ²⁺ flux in CRTh2 expressing cells by anti-CRTh2 antibodies is also provided. An exemplary test can include monitoring cells that respond to Ca ²⁺ flux of a ligand (eg, PGD2) using flow cytometry, followed by incubation with an indo-1/AM dye and an anti-CRTh2 monoclonal antibody.

Immunoconjugate

The invention also provides immunoconjugates comprising an anti-CRTh2 antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (eg, protein toxins) An enzymatically active toxin or a fragment thereof of bacterial, fungal, plant or animal origin) or a radioisotope.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more drugs including, but not limited to, maytansinoid (see US) Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); auristatin, such as the monomethyl auristatin drug moiety DE and DF (MMAE and MMAF) (see U.S. Patent No. 5,635,483 and Nos. 5,780,588 and 7,498,298); dolastatin; calicheamicin or a derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al, Cancer Res. 53:3336-3342 (1993); and Lode et al, Cancer Res. 58:2925-2928 (1998)); Anthracycline, such as daunomycin or doxorubicin (see Kratz et al, Current Med. Chem. 13:477-523 (2006); Jeffrey et al, Bioorganic & Med .Chem. Letters 16:358-362 (2006); Torgov et al., B Ioconj. Chem. 16:717-721 (2005); Nagy et al, Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al, Bioorg. & Med. Chem. Letters 12: 1529 -1532 (2002); King et al, J. Med. Chem. 45: 4336-4343 (2002); and US Patent No. 6,630, 579); methotrexate; vindesine; Taxane, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecene; And CC1065.

In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or a fragment thereof, including but not limited to, a diphtheria A chain, a diphtheria toxin Binding active fragment, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, Acacia toxin A chain, Lycium barbarum toxin A chain, α-Argeuticococcus, tung tree Aleurites fordii) protein, caryophyllin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, diarrhea, croton toxin, sapaonaria officinalis inhibitor , white toxin, mitogellin, restrictocin, phenomycin, enomycin, and crescent toxin.

In another embodiment, an immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioisotopes are available for the production of radioactive conjugates. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu. When the conjugate is used for detection, it may comprise a radioactive atom for scintillation studies, such as tc99m or I123; or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), For example, iodine-123 (again), iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, cesium, manganese or iron.

Conjugates of antibodies to cytotoxic agents can be prepared using a variety of bifunctional protein couplers, such as 3-(2-pyridyldithio)propionic acid N-succinimide (SPDP), 4-(N- Maleidinylmethyl)cyclohexane-1-carboxylic acid amber sulfoxide (SMCC), iminothiolane (IT), bifunctional derivative of imidate (eg hexamethylene dioxime) Imine dimethyl ester HCl), active esters (eg diammonium iodide suberate), aldehydes (eg glutaraldehyde), bis-azido compounds (eg bis(p-azidobenzylidene) Hexamethylenediamine), bis-diazo derivatives (eg bis-(p-diazobenzylidene)-ethylenediamine), diisocyanates (eg toluene 2,6-diisocyanate) and bis-active fluorine compounds (eg 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science , 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylidamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for the coupling of radionucleotides to antibodies. See WO94/11026. A linker can be a "cleavable linker" that promotes the release of a cytotoxic drug in a cell. For example, an acid labile linker, a peptidase sensitive linker, a photolabile linker, a dimethyl linker or a linker containing a disulfide can be used (Chari et al, Cancer Res. 52: 127-131 ( 1992); U.S. Patent No. 5,208,020).

The immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared using the following crosslinker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB ((4-vinyl) The above reagents are commercially available (for example, from Pierce Biotechnology, Inc., Rockford, IL., USA).

Method and composition for diagnosis and detection

In certain embodiments, any of the anti-CRTh2 antibodies provided herein can be used to detect the presence of CRTh2 in a biological sample. The term "detection" as used herein encompasses quantitative or qualitative testing. In certain embodiments, the biological sample comprises cells or tissues, such as Th2 cells, mast cells, eosinophils, basophilic spheres, or congenital type 2 (IT2) cells.

In one embodiment, an anti-CRTh2 antibody for use in a diagnostic or detection method is provided. In yet another aspect, a method of detecting the presence of CRTh2 in a biological sample is provided. In certain embodiments, the methods comprise contacting a biological sample with an anti-CRTh2 antibody described herein under conditions that permit anti-CRTh2 antibody binding to CRTh2, and detecting whether a complex is formed between the anti-CRTh2 antibody and CRTh2. This method can be an in vitro or in vivo method. In one embodiment, an anti-CRTh2 antibody is used to select an individual suitable for therapy with an anti-CRTh2 antibody, for example, wherein the CRTh2 line is used to select a biomarker for a patient.

Exemplary conditions that can be diagnosed using the antibodies of the invention include asthma, granule-poll-type asthma, atopic dermatitis, allergic rhinitis, acute or chronic airway hypersensitivity, eosinophilic syndrome, eosinophilic esophagitis , Qiu-Shi's syndrome, idiopathic pulmonary fibrosis, inflammation associated with cytokines, inflammation or malignancy associated with CRTh2 expressing cells, chronic idiopathic urticaria, chronic spontaneous urticaria, physical Urticaria (including cold urticaria and stress urticaria), bullous pemphigoid, sputum, food allergies and allergic bronchopulmonary bacillary disease with or without cystic fibrosis (ABPA) ).

In certain embodiments, a labeled anti-CRTh2 antibody is provided. Labels include, but are not limited to, directly detected labels or moieties (eg, fluorescent labels, chromogenic labels, electron density labels, chemiluminescent labels, and radioactive labels) and portions that are indirectly detected via, for example, enzymatic reactions or molecular interactions (eg, For example, an enzyme or a ligand). Exemplary labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores (eg, rare earth chelates or fluorescent yellow and its derivatives), rhodamine (rhodamine) And its derivatives, dansyl, umbelliferone, luciferase (eg, firefly luciferase and bacterial luciferase) (US Patent No. 4,737,456), luciferin, 2,3 - Dihydropyridazine dione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase (eg, glucose oxidase, galactose oxidation) Enzymes and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase, which use hydrogen peroxide to oxidize dyes such as HRP, lactoperoxidase or microperoxidase Enzyme coupling of the precursor), biotin/avidin, spin labeling, phage labeling, stable free radicals, and the like.

Medical formulation

The pharmaceutical formulation of an anti-CRTh2 antibody described herein is by mixing the antibody of the desired purity with one or more optional pharmaceutically acceptable carriers ( Remington's Pharmaceutical Sciences , 16th Ed., Osol, A. Ed. (1980) ) is prepared as a lyophilized formulation or as an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methyl sulfide Aminic acid; preservative (eg octadecyl dimethyl benzyl ammonium chloride; hexamethylene diammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzene Methanol; alkyl p-hydroxybenzoate, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; Low molecular weight (less than about 10 residues) polypeptide; protein, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidone; amino acid, such as glycine, glutamine , aspartame, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol , trehalose or sorbitol; salt-forming counterions, examples Such as sodium; metal complexes (such as Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further comprise an interstitial drug dispersing agent, such as a soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, a human soluble PH-20 hyaluronidase glycoprotein, for example rHuPH20 (HYLENEX ^® , Baxter International). Certain example HASEGPs and methods of use (including rHuPH20) are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006/044908, the latter formulation comprising a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient for the particular indication being treated, preferably, such that they have additional active agents that do not adversely affect each other. For example, it may be desirable to further provide, but is not limited to, an IL4 inhibitor (eg, AER-001, IL4/IL13 trap or anti-IL4 antibody), an IL5 inhibitor (eg, mepolizumab ( Mepolizumab), CAS No.196078-29-2; resilizumab or another anti-IL5 antibody), an IL9 inhibitor (eg, MEDI-528 or another anti-IL9 antibody), an IL13 inhibitor (eg , IMA-026, IMA-638 (also known as anrukinzumab, INN No. 910649-32-0; QAX-576; IL4/IL13 trap), tralokinumab (tralokinumab) Also known as CAT-354, CAS No. 1044515-88-9); AER-001, ABT-308 (also known as humanized 13C5.5 antibody) or another anti-IL13 antibody), anti-IL17 antibody, anti-IL25 antibody , anti-IL33 antibody, anti-TSLP antibody, anti-OX40L antibody, anti-OX40 antibody, IL-4-receptor alpha inhibitor (for example, AMG-317, AIR-645 or another anti-IL4Ra antibody), anti-IL5Ra antibody, anti-17RA Antibody or anti-CCR4 antibody. The active ingredients are suitably present in combination in amounts effective for the purpose to be achieved.

The active ingredient may also be separately trapped in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules), glial drugs Delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences , 16th Ed., Osol, A. Ed. (1980).

Sustained release formulations can also be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules.

Formulations intended for in vivo administration are generally sterile. Sterility can be easily achieved by, for example, filtration using a sterile filtration membrane.

Treatment method and composition

Any of the anti-CRTh2 antibodies provided herein can be used in a method of treatment.

In one aspect, an anti-CRTh2 antibody for use as a medicament is provided. In yet another aspect, an anti-CRTh2 antibody for use in treating a condition mediated by CRTh2 is provided. In certain embodiments, an anti-CRTh2 antibody for use in a method of treatment is provided. In certain embodiments, the invention provides an anti-CRTh2 antibody for use in a method of treating an individual having a condition mediated by CRTh2, the method comprising administering to the individual an effective amount of an anti-CRTh2 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as described below. In some embodiments, the disorder is selected from the group consisting of asthma, granule-deficient asthma, atopic dermatitis, allergic rhinitis, acute or chronic airway hypersensitivity, eosinophilic syndrome, eosinophilic Esophagitis, mound-Shih's syndrome, idiopathic pulmonary fibrosis, inflammation associated with cytokines, inflammation or malignancy associated with CRTh2 expressing cells, chronic idiopathic urticaria, chronic spontaneous urticaria, Physical urticaria (including cold urticaria and stress urticaria), bullous pemphigoid, nasal sputum, food allergies, and allergic bronchopulmonary bacillary disease with or without cystic fibrosis (ABPA). In other embodiments, the anti-CRTh2 antibodies provided herein are used to deplete CRTh2 expressing cells in an individual (eg, Th2 cells, mast cells, eosinophils, basophils, and/or congenital type 2 (IT2)) Cells) or reduce one or more cytokines, enzymes, or other inflammatory mediators in an individual (eg, IL-4, IL-5, IL-9, IL-13, IL-17, histamine, neutral protease, and/or Or the content of leukotrienes. In some embodiments, one or more cytokines produced by at least one of the following cell types are reduced: Th2 fine Cells, mast cells, eosinophils, basophils or congenital type 2 (IT2) cells. In certain embodiments, the invention provides for the consumption of CRTh2 expressing cells (eg, Th2 cells, mast cells, eosinophils, basophilic spheres, and/or congenital type 2 (IT2) cells) in an individual and/or Or reducing one or more cytokines, enzymes, or other inflammatory mediators in an individual (eg, IL-4, IL-5, IL-9, IL-13, IL-17, histamine, neutral protease, and/or white) An anti-CRTh2 antibody in a method of the content of a triene), the method comprising administering to the individual an effective amount of an anti-CRTh2 antibody to deplete CRTh2 expressing cells and/or reducing one or more cytokines. The "individual" of any of the above embodiments is preferably human.

In another aspect, the invention provides the use of an anti-CRTh2 antibody in the manufacture or preparation of a medicament. In one embodiment, the agent is for the treatment of a CRTh2-mediated condition. In yet another embodiment, the agent is for use in a method of treating a CRTh2-mediated condition, the method comprising administering to the individual having the condition an effective amount of the agent. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as described below. In some embodiments, the condition is selected from the group consisting of asthma, granule-pollution asthma, atopic dermatitis, allergic rhinitis, acute or chronic airway hypersensitivity, eosinophilia syndrome, eosinophilic ball Sexual esophagitis, mound-Shih's syndrome, idiopathic pulmonary fibrosis, inflammation associated with cytokines, inflammation or malignancy associated with CRTh2 expressing cells, chronic idiopathic urticaria, chronic spontaneous urticaria , physical urticaria (including cold urticaria and stress urticaria), bullous pemphigoid, nasal sputum, food allergies and allergic bronchopulmonary bacteria with or without cystic fibrosis Disease (ABPA). In yet another embodiment, the agent is for consuming a CRTh2 expressing cell (eg, a Th2 cell, a mast cell, an eosinophilic ball, a basophilic ball, and/or a congenital type 2 (IT2) cell) in an individual and/or Decreasing one or more cytokines, enzymes, or other inflammatory mediators in an individual (eg, IL-4, IL-5, IL-9, IL-13, IL-17, histamine, neutral protease, and/or white three) The content of alkene). In yet another embodiment, the agent is for consuming a CRTh2 expressing cell in an individual (eg, Th2 cells, mast cells, eosinophils, basophilic balls, and/or Or congenital type 2 (IT2) cells) and/or reduce one or more cytokines, enzymes or other inflammatory mediators in an individual (eg, IL-4, IL-5, IL-9, IL-13, IL-) 17. A method of administering a content of histamine, neutral protease and/or leukotriene), the method comprising administering to the individual an effective amount of an agent to consume CRTh2 expressing cells and/or to reduce one or more cytokines. The "individual" of any of the above embodiments may be a human.

In yet another aspect, the invention provides methods of treating a CRTh2-mediated condition. In one embodiment, the method comprises administering to an individual having the disorder an effective amount of an anti-CRTh2 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. In some embodiments, the condition is selected from the group consisting of asthma, granule-pollution asthma, atopic dermatitis, allergic rhinitis, acute or chronic airway hypersensitivity, eosinophilia syndrome, eosinophilic ball Sexual esophagitis, mound-Shih's syndrome, idiopathic pulmonary fibrosis, inflammation associated with cytokines, inflammation or malignancy associated with CRTh2 expressing cells, chronic idiopathic urticaria, chronic spontaneous urticaria , physical urticaria (including cold urticaria and stress urticaria), bullous pemphigoid, nasal sputum, food allergies and allergic bronchopulmonary bacteria with or without cystic fibrosis Disease (ABPA). The "individual" of any of the above embodiments may be a human.

In yet another aspect, the invention provides for the consumption of CRTh2 expressing cells (eg, Th2 cells, mast cells, eosinophils, basophilic spheres, and/or congenital type 2 (IT2) cells) in an individual and/or Or reducing one or more cytokines, enzymes, or other inflammatory mediators in an individual (eg, IL-4, IL-5, IL-9, IL-13, IL-17, histamine, neutral protease, and/or white) A method of the content of a triene). In one embodiment, the method comprises administering to the individual an effective amount of an anti-CRTh2 antibody to deplete CRTh2 expressing cells and/or reducing one or more cytokines. In one embodiment, the "individual" is a human. In some embodiments, the individual has a condition selected from the group consisting of: asthma, granule-poll-type asthma, atopic dermatitis, allergic rhinitis, acute or chronic airway hypersensitivity, eosinophilia Group, eosinophilic esophagitis, mound-Shih's syndrome, idiopathic pulmonary fibrosis, inflammation associated with cytokines, inflammation or malignancy associated with CRTh2 expressing cells, chronic idiopathic urticaria, Chronic spontaneous urticaria, physical urticaria (including cold urticaria and stress urticaria), bullous pemphigoid, nasal sputum, food allergies and allergies with or without cystic fibrosis Bronchopulmonary bacillary disease (ABPA).

In certain embodiments, the methods described herein can be used to treat an individual having asthma, wherein the system is eosinophilic positive (EIP) as defined in US 2012/0156194. In certain embodiments, the methods described herein can be used to treat an individual having asthma, wherein the system is eosinophilic negative (EIN) as defined in US 2012/0156194. See also DF Choy et al, J Immunol. 186(3): 1861-9 (2011); Arron et al. (2013) Adv Pharmacol 66: 1-49; and G. Jia et al, J Allergy Clin Immunol. 3): 647-654 (2012).

In certain embodiments, a patient suffering from asthma exhibits a high level of total serum or plasma periostin. In certain embodiments, an EIP patient is a patient whose serum or plasma periostin content has been determined, wherein the serum or plasma periostin content is equal to or greater than the median or mean serum or plasma periostin content of the patient population (also referred to as For high periosteal proteins). In certain embodiments, a patient who has determined serum or plasma periostin content using, for example, an ELISA or sandwich immunoassay as described herein will have a total periostin content (eosinophil positive) of 20 ng/m or greater. According to certain embodiments, the total periostin content in serum or plasma of a patient for EIP can be selected from the group consisting of: 21 ng/ml or higher, 22 ng/ml or higher, 23 ng/ml or higher, 24 ng. /ml or higher, 25 ng/ml or higher, 26 ng/ml or higher, 27 ng/ml or higher, 28 ng/ml or higher, 29 ng/ml or higher, 30 ng/ml or higher, 31 ng/ Ml or higher, 32 ng/ml or higher, 33 ng/ml or higher, 34 ng/ml or higher, 35 ng/ml or higher, 36 ng/ml or higher, 37 ng/ml or higher, 38 ng/ml Or higher, 39 ng/ml or higher, 40 ng/ml or higher, 41 ng/ml or higher, 42 ng/ml or higher, 43 ng/ml or higher, 44 ng/ml Or higher, 45 ng/ml or higher, 46 ng/ml or higher, 47 ng/ml or higher, 48 ng/ml or higher, 49 ng/ml or higher, 50 ng/ml or higher, 51 ng/ml or Higher, 52 ng/ml or higher, 53 ng/ml or higher, 54 ng/ml or higher, 55 ng/ml or higher, 56 ng/ml or higher, 57 ng/ml or higher, 58 ng/ml or more. High, 59 ng/ml or higher, 60 ng/ml or higher, 61 ng/ml or higher, 62 ng/ml or higher, 63 ng/ml or higher, 64 ng/ml or higher, 65 ng/ml or higher 66 ng/ml or higher, 67 ng/ml or higher, 68 ng/ml or higher, 69 ng/ml or higher, and 70 ng/ml or higher.

In certain embodiments, a patient suffering from asthma exhibits a low level of total serum or plasma periostin. In certain embodiments, an EIN patient refers to a patient who has been tested for serum or plasma periostin content, wherein the serum or plasma periostin content is less than 20 ng/ml.

It should be appreciated that the EIP status represents the condition of the patient and does not depend on the type of analysis used to determine the status. Therefore, other eosinophilic inflammatory diagnostic assays (including other periostin assays, such as ELISA assays and ELECSYS® periosteal protein assays shown in US 2012/0156194) can be used or developed to test eosinophilic inflammatory states and measure total periostin content. See also, Jia et al., 2012, J. Allergy Clin. Immunol. 130: 647-654 and US 2012/0156194, the entire contents of which are hereby incorporated by reference.

The term "total periostin" as used herein refers to at least the isoforms 1, 2, 3 and 4 of periostin. According to this technique, human periostin isoforms 1, 2, 3, and 4 contain the following amino acid sequences, respectively, according to the NCBI database: NP_006466 (SEQ ID NO: 87); NP_001129406 (SEQ ID NO: 88), NP_001129407 (SEQ ID NO: 89) and NP_001129408 (SEQ ID NO: 90), and isoform 5 has been partially sequenced. Isoform 5 comprises the amino acid sequence of SEQ ID NO:91. In one embodiment, the isoform of the periostin is a human periostin. In yet another embodiment, the term total periostin divided by isoforms 1-4 includes isoforms 5 of human periostin. In another embodiment, the total periostin is total serum periostin or total plasma periostin (ie, total periostin from a serum sample obtained from whole blood or a plasma sample obtained from whole blood, respectively, the whole blood line Self-patient Got).

In some embodiments, an anti-CRTh2 antibody administered to an individual consumes CRTh2 expressing cells in the individual. In some embodiments, the antibody consumes CRTh2 expressing cells from lung tissue and/or bronchoalveolar lavage fluid. In some embodiments, at least one type of CRTh2 expression cell (eg, from the lung) in the individual is consumed at least about 50%, 60%, 70%, 80%, 85%, 90 compared to the baseline prior to administration of the antibody. Any of %, 95%, and 100%. In some embodiments, at least one type of cytokine Th2 producing cell (eg, from the lung) in the individual is consumed at least about 50%, 60%, 70%, 80%, 85% compared to the baseline prior to administration of the antibody. , 90%, 95% and 100%. As used herein, "baseline" refers to the amount prior to administration of an anti-CRTh2 antibody described herein to an individual. The amount of CRTh2 expressing cells before and after administration of the antibody can be tested using methods known in the art and described herein.

In yet another aspect, the invention provides a pharmaceutical formulation comprising any of the anti-CRTh2 antibodies provided herein for use, for example, in any of the above methods of treatment. In one embodiment, a pharmaceutical formulation comprises any of the anti-CRTh2 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the anti-CRTh2 antibodies provided herein and at least one additional therapeutic agent, for example as described below.

The antibodies of the invention may be used in therapy alone or in combination with other agents. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an inhaled corticosteroid, a short acting beta 2 agonist, a long acting beta 2 agonist, a long acting muscarinic agonist, a leukotriene receptor antagonist, a mast cell inhibitor (eg, , Comarine), CRTh2 small molecule inhibitors or combinations thereof.

The combination therapies mentioned above encompass the combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and administered separately (in which case the administration of the antibodies of the invention may be Prior to, concurrently with, and/or after administration of additional therapeutic agents and/or adjuvants.

The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and (if desired for topical treatment) intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on the length of administration. This article covers a variety of dosing schedules including, but not limited to, single or multiple doses, bolus doses, and pulse infusions at different time points.

The antibodies of the invention should be formulated, metered and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause, the delivery site of the agent, the method of administration, the schedule of administration, and the medical practitioner. Know other factors. The antibody need not (but optionally) be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. Such agents are typically administered in the same dosages and in the administration routes described herein, or from about 1% to 99% of the dosages described herein, or in any dosage or any route that is empirically/clinically determined to be suitable.

For the prevention or treatment of a disease, the appropriate dose of the antibody of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and duration of the disease, and the administration The anti-system is used for prophylactic or therapeutic purposes, prior treatment, clinical history of the patient and response to antibodies, and judgment of the attending physician. The anti-system is administered to the patient in a suitable manner, either at one time or through a series of treatments. Depending on the type and severity of the disease, from about 1 [mu]g/kg to 15 mg/kg (eg, 0.1 mg/kg to 10 mg/kg) of the antibody can be the initial candidate dose for administration to the patient, whether by, for example, one or more times. Individually administered, or by continuous infusion. Depending on the above factors, a typical daily dose may be between about 1 [mu]g/kg and 100 mg/kg or higher. For repeated administrations over several days or longer, depending on the condition, treatment will generally continue until the desired repression of the symptoms of the disease occurs. An exemplary dosage of an antibody can range from about 0.05 mg/kg to about 10 mg/kg. Therefore, One or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or a combination thereof) are administered to the patient. Such doses can be administered intermittently, for example once a week or once every three weeks (eg, such that the patient receives from about 2 to about 20 or, for example, about 6 doses of the antibody). A higher loading dose can be administered at the beginning, followed by one or more lower doses. However, other dosage regimens can be used. The progress of this therapy can be easily monitored by conventional techniques and analysis.

It will be appreciated that the immunoconjugate of the invention may be used in place of or in combination with an anti-CRTh2 antibody to perform any of the above formulations or methods of treatment.

Products and kits

In another aspect of the invention, an article or kit comprising one or more anti-CRTh2 antibodies useful for treating, preventing, and/or diagnosing the conditions described above is provided. The article or kit can further comprise a container and a label or package insert located on or against the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The containers may be formed from a variety of materials such as glass or plastic. The container contains a composition effective to treat, prevent, and/or diagnose a condition when combined with itself or in combination with another composition, and may have a sterile access port (eg, the container may be an intravenous solution bag or have a hypodermic needle) a vial pierced by a stopper). At least one active agent in the composition is an antibody of the invention. A marker or package insert indicates that the composition is used to treat a selected condition. Additionally, the article or kit can comprise (a) a first container comprising the composition, wherein the composition comprises an antibody of the invention; and (b) a second container comprising the composition, wherein the composition comprises another Cytotoxic agent or opposite therapeutic agent. The article or kit of this embodiment of the invention may further comprise a package insert indicating that the composition is useful for treating a particular condition. Alternatively or additionally, the article or kit may additionally comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution ( Ringer's solution) and dextrose solution. It may additionally include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and bets. Projector.

It will be understood that any of the above articles or kits may comprise an immunoconjugate of the invention in place of or in combination with an anti-CRTh2 antibody.

Instance

The following are examples of the methods and compositions of the present invention. It will be appreciated that various other embodiments may be practiced in light of the general description provided above.

Example 1. Generation of murine anti-human CRTh2 antibody Materials and methods Colonization and cell line

Rhesus monkey and cynomolgus CRTh2 cDNA were obtained by RT-PCR from total RNA extracted from rhesus monkeys and cynomolgus monkey blood and colonized in mammalian expression vector with amine-based end Flag tag, gD tag or unlabeled In the pRK5 vector. Human full-length cDNA from Origene (Gene Bank NM_004778) was cloned into vector pRK5 with an amine-terminal Flag tag, gD tag or no tag. Upon sequence confirmation, the CRTh2 pure line contains alanine instead of proline at position 204, as indicated in gene bank NM_004778 (eg, SEQ ID NO: 84 with V204A substitution relative to the gene bank reference sequence).

The CRTh2-containing plasmid was transfected into 293 cells using Fugene 6 (Roche) and used with anti-Flag antibody (pure line M2, Sigma), anti-gD Ab (pure line 952, Genentech) or rat anti-human CRTh2 The specific anti-CRTh2 Ab of CRTh2 antibody BM16 (BD Pharmingen) confirms the surface appearance of labeled or unlabeled CRTh2. The CRTh2-containing plasmid was also introduced into 300.19 cells (mouse pre-B cell line) by electroporation, and the surface CRTh2 expression was confirmed by the Flag tag expression. The expression of CRTh2 on the surface of 300.19 cells was also determined by an anti-CRTh2 monoclonal antibody against human CRTh2 including BD Pharmingen.

Anti-human CRTh2 antibody production

For the generation of anti-human CRTh2 antibodies, Balb/c mice (Charles River (Charles) River)) Immunize with one of two methods: DNA immunization and cellular immunity. For DNA immunization, Balb/c mice were immunized weekly by hydraulic injection of 50 ug of human CRTh2 DNA in pRK5 vector plus mouse Flt3-L and GM-CSF as adjuvants. For cellular immunity, Balb/c mice (Charles River, Hollister, CA) were injected intraperitoneally by intraperitoneal injection of 5 million 300.19 cells stably transfected with human CRTh2 in PBS via ip injection twice a week. Immunity. Mice received 10 doses followed by pre-fusion boost with 20 million cells i.v. together with 40 million cells i.p. 3 days prior to fusion.

Fusion tumors are generated by standard methods. Splenocytes were fused with X63-Ag8.653 mouse myeloma cells (American Type Culture Collection, Rockville, MD) via electrofusion (BTX, Hawthorne, NY) and at 37 ° C, 7% Dulbecco's Modified Eagle's Medium (DMEM; Lonza, Basel, Switzerland) was incubated with CO2 supplemented with: 10% fetal bovine serum (FBS), 4.5 g/L glucose, 25 mM HEPES, 0.15 mg/ml oxaloacetate, 100 μg/ml pyruvate, 0.2 U/ml insulin, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (penicillin-streptomycin, Invitrogen , Carlsbad, CA), NCTC-109 (Lonza), NEAA (Invitrogen), then plated in 96-well plates as described with 5.7 μM azoserine and 100 μM hypoxanthine (HA, Sigma-Aldrich, St .Louis, MO) in the medium. The cells were cultured for 10 days, followed by ELISA and FACS analysis. Cells from the wells and demonstrating expression of mouse IgG by FACS and showing strong specific binding were expanded and subcloned by limiting dilution. The final pure line expansion confirming the strongest FACS binding after the second round of sub-colonization was used for large scale production in bioreactors (Integra Biosciences, Chur, Switzerland). The supernatant was then purified by protein A affinity chromatography as previously described (Hongo et al, Hybridoma 19: 303, 2000). Purified antibodies from fusion tumors were screened by flow cytometry for the ability to bind to human CRTh2 expressed on 293 cells or 300.19 cells. The binding reactivity to basophilic and eosinophils from human peripheral blood was also tested. The light and heavy chains of the pure lines 19A2, 8B1, 31A5 and 3C12 were subcloned into the pRK5 vector. All heavy chains were selected to contain the mouse IgG2a Fc region. Anti-CRTh2 antibodies were generated in CHO cells using standard procedures. Afucosylated 19A2 and 8B1 antibodies were generated from the FUT8 ^-/- CHO cell line.

Flow Cytometry

Human whole blood was obtained from healthy donors and peripheral blood mononuclear spheres (PBMC) were used for the staining procedure after erythrocyte lysis using EL buffer (Qiagen). Blood cells were incubated with A467-conjugated anti-CRTh2 antibody at various concentrations or with anti-CRTh2 antibody plus secondary anti-mouse IgG-PE antibody (Jackson ImmunoResearch Laboratory). The anti-system used to stain the white blood cell population is as follows: FITC-anti-human CD15, CD16, PerCP-anti-human HLADR and CD4, APC-anti-human CD123, CXCR3, CD14, BDCA1, biotin-anti-human CCR6 and PE-anti-human CCR4 . The Ab used was purchased from BD Pharmingen. To determine the expression of CRTh2 on regulatory T cells, CD4+CD25+ T cells were enriched from human PBMC by MACS isolation (Miltenyi Biotec), surface staining with anti-CRTh2 (antibody BM16), followed by anti-FoxP3 (BD Bioscience) Perform intracellular staining. To assess the performance of CRTh2 on human mast cells, StemPro-34 SFM Complete Medium (Gibco) with 200 ng/mL rhIL-6, 100 ng/mL rhSCF (PeproTech) and 30 ng/mL rhIL-3 (R&D system) Fresh human bone marrow CD34+ cells (AllCells) were cultured for 3-4 weeks to generate mast cells. Mast cells were stained with anti-CD117, anti-CD123 and anti-FceRI (BD Bioscience). To determine the expression of human CRTh2 on Th2 cells in human CRTh2.Bac.Tg mice, 50 ug of papain in 50 ul PBS was injected into the right hind footpad of mice, and after 3 days, axillary lymph node cells were collected and anti-mCD4 was used. -PerCP, anti-mCD44-FITC and BM16-A647 staining. To examine the performance of human CRTh2 on congenital T-helper (IT) 2 cells in human CRTh2.Bac.Tg mice, 50 ug of mouse IL-17E in the pRK5 vector was hydrodynamically injected into the tail vein. . After 3 days, mesenteric lymph node cells were collected and treated with anti-mCD117-PE, BM16-A647, iodide The propidium and lineage markers (FITC markers: CD3, CD4, CD8, B220, FceRI, CD11c, Gr1, NK1.1, F4/80, DX5 and PerCP markers: CCR3) were stained. FITC-anti-human CD15, CD16, PerCP-anti-human HLADR and CD4, APC-anti-human CD123, CXCR3, CD14, BDCA1, biotin-anti-human CCR6 and PE-anti-human CCR4 lines were purchased from BD Pharmingen. Peripheral blood mononuclear spheres (PBMC) were used for staining procedures after cynomolgus monkeys and rhesus monkey blood were obtained from healthy monkeys and after red blood cell lysis was performed using EL buffer (Qiagen). Blood cells were incubated with A467-conjugated anti-CRTh2 antibodies at various concentrations. The anti-system used to stain the white blood cell population was as follows: FITC-anti-human CD123 (BD Pharmingen), PE-anti-human CD125 (BD Pharmingen) and PerCP-eFluor710 anti-human FceRI (eBioscience). Samples were captured on a FACSCalibur flow cytometer using CellQuest Pro software (BD Biociences) and data analysis was performed using Flowjo (Tree Star, Inc).

Separation of CRTh2 + memory CD4 + T cells and quantification of cytokine production

Human PBMCs from specific donors were isolated by non-touch memory CD4+ T cells by MACS isolation (Miltenyi Biotec), followed by CD45RO-FITC, CD4-PerCP (BD Pharmingen) and BM16-PE (Miltenyi Biotec) antibodies at 37 Dye for 20 minutes at °C. CRTh2+CD45RO+CD4+ and CRTh2-CD45RO+CD4+ memory T cells were sorted by a FacsAria sorter (BD). The purity of CRTh2+ and CRTh2-memory CD4+ T cells was higher than 98%. The same number of sorted cells were stimulated with a 10 ug/mL plate in combination with anti-hCD3 mAb and 1 ug/mL soluble anti-hCD28 for 48 hours at 37 °C. The supernatant was collected and assayed for IL-4, IL-5, IL-9, IL-13, IL-17A, TNFα, IFNγ and GM-CSF using human Bio-Plex (Bio-Rad) antibody-immobilized beads. The Luminex 100 instrument (Luminex) reads the plates according to the manufacturer's protocol.

Radioligand cell binding assay (Schachad analysis)

Cells radioligand equilibrium dissociation assay showed anti-CRTH2 antibody binding of recombinant cells CRTh2 receptor dissociation constant (K _D) binding assays. The anti-CRTh2 antibody was iodinated using the Iodogen method and the specific activity of the labeled antibody was in the range of 19-22 μCI/μg for the Fab antibody and 10-14 μCI/μg for the IgG antibody. Cells expressing the CRTh2 receptor were incubated for 2 hours at room temperature with a fixed concentration of the iodinated anti-CRTh2 antibody and a combination of increasing concentrations of unlabeled anti-CRTh2 antibody and including a zero addition buffer only sample. After 2 hours of incubation, the competition reaction was transferred to a Millipore Multiscreen filter plate and washed 4 times with binding buffer to isolate the free iodinated antibody and bound iodinated antibody. The filter was counted on a Wallac Wizard 1470 gamma counter. Binding data was evaluated using NewLigand software (Genentech) using the fitting algorithm of Munson and Rodbard to determine the binding affinity of anti-CRTh2 antibodies (Munson and Rodbard, Anal. Biochem, 1980; 107: 220-239).

Epitope mapping

Purified mouse anti-CRTh2 monoclonal antibody 19A2 and rat anti-CRTh2 monoclonal antibody BM16 were biotinylated using the EZ-Link Sulfo-NHS-Biotin kit (Pierce/Thermo-Fisher, Rockford, IL). Activity was confirmed by FACS titration of stably transfected 293 cells with human CRTh2 or rhesus CRTh2. 50 ul of transfected 293 cells were added to a 96-well U-bottom plate (BD Falcon, Franklin Lakes, NJ) at a concentration of 10 million cells/ml and suspended in PBS containing 1% FBS, followed by 20 ug/ 50 μl of unlabeled antibody was added at a concentration of ml, and the plate was incubated at 4 ° C for 30 minutes. The biotinylated antibody was added to the plate at a concentration of 2 ug/ml (19A2 and BM16) as determined by a previous FACS titration test, and the plate was incubated at 4 ° C for 30 minutes. The cells were washed twice with centrifugation to sediment the cells, followed by the addition of 200 μl of PBS containing 1% FBS. The cells were then incubated with phycoerythrin-coupled streptavidin (Zymed/Life Technologies, Grand Island, NY) for 30 minutes at 4 °C. The cells were washed, fixed in PBS containing 1% formalin, and analyzed by flow cytometry on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

Human T cell polarization

Untreated untreated CD4+ T cells were isolated from healthy donor PBMCs by MACS isolation (Miltenyi Biotec). The cells were in complete DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 50 uM 2-ME, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 ug/mL streptomycin and 1 mM non-essential amino acid. Incubate in the presence of 10 ug/mL plate-bound anti-CD3 mAb and 1 ug/mL soluble anti-CD28 mAb (BD Biociences). The polarization conditions of human Th subsets are as follows: Th1: 10 ng/mL rhIL12 and 2 ug/mL anti-hIL-4; Th2: 20 ng/mL rhIL-4 (R&D System), 2 ug/mL anti-hIL-12 and 5 ug/mL anti- hIFNγ (BD Biosciences). Two rounds of polarization were performed, each consisting of an activation period followed by a rest period and a second restimulation was performed in the presence of 1 ug/mL plate-bound anti-CD3 mAb and 1 ug/ml soluble anti-CD28 mAb.

Calcium mobilization analysis

In vitro polarized Th2 cells were incubated with 5 μM indo-1/AM and 0.2% pluronic F127 (Molecular Probe) at 37 ° C for 30 minutes, washed and then treated with anti-CCR6, anti-CCR4, anti-CD4 The anti-CXCR3 mAb was stained for 15 analysis at 37 ° C; after washing, the cells were incubated with 1 uM anti-CRTh2 mAb or isotype control antibody for 30 minutes at 37 ° C, and then stimulated with 100 nM PGD2 (Sigma). Calcium release was monitored by flow cytometry.

CRTh2 functional cAMP blocking analysis

Anti CRTh ₂ recombinant cell lines _{^{cAMP Hunter TM CHO-K 1 CRTh}} 2 Gi; After incubation with (DiscoveRx Fremont, CA), the forskolin and by PGD _2; the presence (Sigma St.Louis, MO) under the measuring cAMP levels of anti CRTh2 cells PGD ₂ -CRTh ₂ antibody in the signaling blocking activity. Briefly, cells were grown overnight at 10,000 cells/well in 384 well tissue culture plates (Corning Cat # 3707; Corning, NY). After the medium was removed, 10 μL of the test antibody (continuous dilution in PBS) was added to each well and incubated at 37 ° C for 30 minutes. Forskolin and PGD _{2 were then} added to each well to reach a final concentration of 12.5 μM and 4 nM, respectively. After incubation for an additional 30 minutes at 37 °C, plates were subjected to cAMP analysis using a HitHunter® cAMP XS+ kit (DiscoveRx) according to the manufacturer's protocol, with Envison (Perkin Elmer; Waltham, MA) as a chemiluminescent reader. The data was then analyzed and the curve was plotted using Prism (GraphPad Software; La Jolla, CA).

Generation of human CRTh2 BAC transgenic mice

A 171 Kb fragment (RPCI human BAC library 11; pure line ID RP11-68H20) containing the human CRTh2 gene in the BAC vector backbone was purchased from Invitrogen. A shorter form of 28 Kb was obtained with the aid of a reorganization project. The 171 Kb or 28 Kb BAC construct was microinjected into fertilized oocytes harvested from C57BL/6 mice. The presence of the human CRTh2 transgene was determined by RT-PCR of mouse tail DNA. Of the 9 identified first-time constructors, 7 gave similar human CRTh2 expression patterns to immune cell types tested by flow cytometry. In these lines, line 85 was confirmed by flow cytometry on mouse basophilic and eosinophilic spheres compared to human CR12 content from human primary basophils and eosinophils from human PBMC. Has similar human CRTh2 expression levels.

Measurement of blood basophilic and eosinophilic anti-CRTh2 antibody consumption in human CRTh2 BAC transgenic mice

Mouse or humanized anti-human CRTh2 antibody or isotype control antibody (mIgG1: anti-gp120 antibody; mIgG2a: anti-porcine antibody; hIgG1: anti-gD antibody) was intravenously injected at the indicated dose for 6-8 weeks on day 0 In human CRTh2 BAC tg mice. Ocular blood was obtained after 3, 6 or 7 days to analyze the number of basophilic and eosinophils by flow cytometry as indicated. Alternatively, a group of mice are sacrificed on day 2, day 3, 7 or day 14 and blood, spleen and bone marrow are harvested and processed for calculation of eosinophilic and basophilic balls by flow cytometry. . Red blood cells were lysed using EL buffer (Qiagen). White blood cells, spleen cells or bone marrow cells were stained with anti-CD123-FITC, anti-FcεRI-PE and anti-CCR3-PerCP to detect basophilic and eosinophils. Absolute cell numbers were determined by flow cytometry using CaliBRITE FITC beads (BD Biosciences).

TNP-OVA-induced lung inflammation in human CRTh2 BAC transgenic mice

Human CRTh2 BAC tg mice were sensitized on day 0 by intraperitoneal injection of 50 ug of TNP-OVA (Biosearch Technologies) in 2 mg of aluminum hydroxide in 100 ul of sterile PBS. Starting on day 35 after sensitization, mice were challenged with nebulized 1% TNP-OVA in PBS for 30 minutes via a nebulizer for 7 consecutive days. Mice were used once daily from day 38 to day 41 for 200 ug of anti-human CRTh2 antibody pure 19A2 mIgG2a (no fucosylation or wt), Fc mutant Ab 19A2 mIgG2a_DANA or anti-porcine control antibody in 100 uL of normal saline. (mIgG2a) intraperitoneal treatment. All mice were euthanized on day 42. The mice were perfused with 20 ml of PBS in the right ventricle to wash the peripheral blood of the lungs and the entire lung was removed for flow cytometry. Blood was collected by cardiac puncture for evaluation by flow cytometry. BAL was collected for cell counting and cytokine analysis. BAL fluid IL-4 and IL-13 cytokine concentrations were determined by ELISA (R&D) according to the manufacturer's protocol. The 19A2 antibody 19A2_DANA in an effector-deficient form was produced by mutating the two residues (D265A, N297A) that abolished the Fcγ receptor binding.

Human SCID model to assess the potential of anti-human CRTh2 Ab to consume Th2 cells

On day -7, human PBMCs were isolated from specific donors with a serum IgE content of 315 ng/mL by white blood cell separation and Ficoll density gradient centrifugation (GE Healthcare). Aliquots of PBMC from the same donor were frozen for subsequent transfer to mice. Untreated untreated CD4+ T helper cells were further isolated from PBMC by using untreated CD4+ T cell isolation kit II (Miltenyi Biotec 130-094-131) to consume non-CD4+ T cells and memory T cells. Purified untreated CD4+ T cells were plated with anti-CD3 at 10 ug/mL (BD 555329) and 1 ug/mL soluble anti-CD28 (BD 555725) under twisting conditions against Th2: 5 ug/mL anti-human IFNg (BD 554698) 2 μg/mL anti-IL12 (BD 554659) and 20 ng/mL recombinant human IL-4 (R&D System 204-IL) were stimulated for 3 days. CD4+ T cells stimulated under Th2 conditions were used for transfer assays in SCID gray-brown mice.

On day 0, SCID gray-brown mice (Charles River) were irradiated with a 3.5 Gy 137 source at a sublethal dose. Will 100ul PBS in the human T-cell via the following intraperitoneal injection of the mixture was transferred to mice: from the same donor of 6 × 10 ⁷ T cells polarized warp and 4 × 10 ⁷ th (as described above) Live previously frozen human PBMC. On day 0 and day 3, 100 ug of anti-human IFNg and 100 ug of anti-human IL-12 antibody in 100 ul PBS were intraperitoneally injected into mice. On day 1, day 2, and day 3, 100 ng of recombinant human IL-4 was intraperitoneally injected into mice. Mice were treated with 200 ug of anti-human CRTh2 antibody (pure line 19A2, no fucosylation) or anti-porcine isotype control antibody in 100 uL PBS on days 0 and 3 prior to cell transfer. All mice were euthanized on day 7 and the spleens were collected. Splenocytes were stimulated in vitro using PdBu (50 ng/mL) and ionomycin (500 ng/mL) for 4.5 hours at 37 ° C for evaluation of intracellular cytokine content by FACS. Cells were surface stained with anti-hCD4 and stained in the same channel using anti-mCD45, anti-mTer119 and anti-hCD19 to exclude these lineage positive cells. The cells were fixed and stained with anti-hIFNg and anti-hIL-4.

Model for IT2 cell consumption

To increase the number of congenital T-helper (IT) 2 cells in human CRTh2.Bac.Tg mice, 50 ug/mouse IL-17E in pRK5 vector was hydrodynamic in 1.6 ml Ringer's solution. The method is injected into the tail vein. After 3 days, mesenteric lymph nodes were collected and stained with anti-mCD117-PE and BM16-A647 by flow cytometry and excluded lineage positive and dead cells (lines: CD3, CD4, CD8, B220, FceRI, CD11c, Gr1, NK1. 1. F4/80, DX5 and CCR3) Determine the percentage and quantity of IT2 cells.

result

CRTh2 is expressed on cells associated with asthma

The expression of CRTh2 on cells from human PBMC or cultured human cells was assessed by flow cytometry using anti-human CRTh2 antibody (pure line BM16) (Fig. 1). CRTh2 selectively expressed in human blood basophilic bulbs, eosinophils, polarized Th2 cells, bone marrow source fertilizer Large cells and congenital T-assisted 2 (IT2) cells, as recently reported (Mjosberg, Nat. Imm. 12(11): 1055-62 (2011)). CRTh2 is not expressed on polarized Th1 cells, neutrophils, dendritic cells, monocytes, and regulatory T cells. No CRTh2 expression was detected on B cells, NK cells, NK T cells, and platelets (data not shown).

CRTh2 cells are associated with Th2 cytokine production

To assess the performance of CRTh2 on T cell subsets associated with Th2 cytokine production, CRTh2+ and CRTh2-memory CD4 T cells were subjected to FACS sorting and stimulated with anti-CD3 and anti-CD28 antibodies to assess Th2 cytokine production. CRTh2+ memory CD4 T cells produced 95% more memory T cell Th2 cytokines than the CRTh2-memory CD4 T cell population (Fig. 2). Other donors tested showed similar results.

Generation and in vitro characterization of anti-human CRTh2 antibody

The anti-human CRTh2 anti-system was generated from Balb/c mice that had been immunized with 300.19 cells expressing human CRTh2 from no adjuvant.

The anti-CRTh2 antibody generated as described herein binds to 293 cells or 300.19 cells that express human CRTh2 in a dose-dependent manner, but does not bind to 293 or 300.19 wild-type cells (Figs. 3A and 3B). The cross-reactivity of anti-human CRTh2 Ab with cyno or rhesus CRTh2 overexpressing in 293 or 300.19 cells was tested. All Ab except for the pure line 19A2 showed no reactivity with cynomolgus monkey or rhesus monkey CRTh2, and the pure lineage 19A2 showed little cross-reactivity with the cynomolgus CRTh2 expressed on 293 cells. Anti-human CRTh2 antibodies were also dose-dependent with primary human basophilic and eosinophils from human whole blood. The candidate anti-human CRTh2 anti-system is based on its ability to bind to human CRTh2 overexpressing 293 cells or 300.19 cells and its relative to primary basophilic and eosinophils from human peripheral blood mononuclear cells (PBMC). Reactivity is chosen (Figure 3). All other antibodies generated from the above immunizations bind to human CRTh2 but do not cross-react with rhesus or cynomolgus CRTh2 (data not shown). Also tested humanized pure line h19A2.v1 and pure line h19A2.v12 with CRTh2 expressed on 293 cells or 300.19 cells and with primary blood Reactivity of phloem and CRTh2 on eosinophils. Similar to 19A2, humanized h19A2.v1 reacts with human CRTh2 expressed on 293 cells (Fig. 3D), 300.19 cells (Fig. 3E), and CRTh2 (Fig. 3F) on primary blood basophilic and eosinophilic spheres, It has minimal cross-reactivity with cynomolgus CRTh2 that is overexpressed on 293 or 300.19 cells. The humanized h19A2.v1 did not react with rhesus CRTh2, which was expressed on 293 or 300.19 cells and the blood basophilic ball of the first rhesus monkey. In contrast, the humanized and genetically engineered antibody h19A2.v12 was expressed in a dose-dependent manner in human, cynomolgus and rhesus CRTh2 and in the first generation of 293 cells (Fig. 3D) or 300.19 cells (Fig. 3E). Human, cynomolgus and rhesus CRTh2 (Fig. 3F) reactions on blood basophilic balls. In addition, antibody h19A2.v12 was also detected in CRTh2 on the first human blood eosinophils.

Homologous competition embodiment of the radiolabeled ligand analysis showed the antibody solution anti-CRTh2 cells and 293 cells on the surface of human CRTh2 300.19 evaluation dissociation constant (K _D). Mouse anti-human CRTh2 Homogenous 19A2 and 8B1 (whole IgG) on expression of human CRTh2 K _D values of the 293 cells were 2nM and 2.6nM. Antibody 19A2 value of K _D 300.19 cells of 10.2 nm (FIG. 4A). It is directly measured K _D value and humanized antibodies h19A2.v12 h19A2.v60 of generated Fab fragments of such antibodies and cells subjected to radioligand binding assay (FIG. 4B). h19A2.v12 and h19A2.v60 (IgG Fab fragments of) human performance to K _D value of 293 CRTh2 cells were 51nM and 56nM. h19A2.v12 and h19A2.v60 (IgG Fab fragments of) performance of cynomolgus CRTh2 cells of 293 K _D values were 152nM and 39nM. Based on these measurements, the relative binding affinity of human cynomolgus CRTh2 to h19A2.12 was within 3 fold and appeared to be equivalent for h19A2.v60 (Fig. 4B).

To obtain human antibodies h19A2.v52 and direct measurement of K _D values h19A2.v46 generated Fab fragments of such antibodies and cells subjected to radioligand binding assay (FIG. 15). h19A2.v52 and h19A2.v46 (IgG Fab fragments of) human performance to K _D value of 293 CRTh2 cells were 13.7nM and 6.4nM. h19A2.v52 and h19A2.v46 (IgG Fab fragments of) performance of cynomolgus CRTh2 cells of 293 K _D values were 21.3nM and 8.6nM.

To assess the blocking function of anti-CRTh2 antibodies, calcium mobilization of ligand prostaglandins (PGD2) by in vitro polarized Th2 cells was examined in the presence of anti-CRTh2 or isotype control antibodies. The calcium flux to PGD2 was completely prevented by pre-incubation of cells with 8B1 and 3C12, while 31A5 showed partial effects. Incubation with the anti-CRTh2 19A2 antibody did not significantly affect CA2+ flux (Figure 5), indicating that the 19A2 is a non-blocking antibody to CRTh2 compared to 8B1 and 3C12, which block the function of CRTh2.

Generation of a transgenic mouse model of human CRTh2

To characterize the in vivo consumption of anti-CRTh2 antibodies, a transgenic mouse model (human CRTh2.Bac.Tg mice) was generated by introducing the human CRTh2 gene on the BAC vector into C57BL/6 fertilized oocytes (Fig. 6A). ). Although the performance of human CRTh2 on blood basophils and eosinophils was confirmed on 7 primary constructors, the expression of hCRTh2 on mouse Th2 cells in the hCRTh2.Bac.tg strain was not detected. A more detailed analysis of the three representative first-builder lines (data not shown). Compared with the first-generation human blood basophils and eosinophils and bone marrow-derived human mast cells (Fig. 6B), the first strain of human CRTh2.Bac.Tg mice was confirmed to be in the blood basophilic ball of mice. The expression levels of human CRTh2 on eosinophils and peritoneal mast cells were similar. Therefore, all of the first constructors, 85 hCRTh2.Bac.Tg mice, were used in subsequent in vivo consumption studies. Furthermore, the first constructor line 85 (Fig. 6B) of human CRTh2 on mouse congenital T-helper (IT) 2 cells appeared to be reduced in content compared to the performance on human IT2 cells from PBMC.

Anti-CRTh2 antibody consumes blood wave basophilic ball and eosinophilic ball in human CRTh2.Bac.Tg mice

To test whether anti-CRTh2 antibodies can consume CRTh2+ basophils and eosinophils in vivo, one dose of 19A2 or 3C12 antibody was administered to CRTh2.Bac.Tg mice as indicated (Fig. 7A). A single dose of 19A2 or 3C12 completely consumed the basophilic and eosinophilic spheres in peripheral blood of human CRTh2.Bac.Tg mice on day 3 after treatment, as by flow As determined by cytology (Fig. 7A). Significant consumption of basophilic and eosinophils was still observed on day 7 after treatment. The 8B1 and 19A2 antibodies also consumed blood-derived basophilic and eosinophilic spheres on day 6 after treatment as assessed by a single dose of antibody (Fig. 7B).

Anti-CRTh2 antibody depletes eosinophilic and basophilic balls in the lung in a TNP-OVA-induced chronic asthma model in human CRTh2.Bac.Tg mice

To assess whether anti-CRTh2 antibodies can deplete CRTh2+ cells in tissues, the effect of anti-CRTh2 antibody treatment was examined in a TNP-OVA-induced chronic asthma model. In the i.p. 200 ug/mouse regimen, 4 doses of antibody 19A2 completely consumed lung eosinophils and basophils, and lung mast cells were also consumed 80% (Figure 8). In addition, the eosinophilic bulb in bronchoalveolar lavage fluid (BALF) was consumed by 100%. In addition, Th2 cytokines IL-4 and IL-13 in bronchoalveolar lavage fluid (BALF) were reduced by 100% and 48%, respectively (Fig. 8B).

Anti-CRTh2 antibody depletes cells producing Th2 cytokines in SCID mice

Since human CRTh2 expression was not detected on Th2 cells (CD4+CD44hi) in human CRTh2.Tg mice, it was not possible to evaluate Th2 cell consumption in human CRTh2.Bac.Tg mice. To evaluate whether anti-CRTh2 antibodies can consume Th2 cytokine-producing cells in vivo, in vitro transduced human Th2 cells were transferred to SCID mice, treated twice weekly with anti-CRTh2 or isotype control Ab, and at the beginning IL-4 producing cells were evaluated after ex vivo stimulation with PMA and ionomycin on day 7 after administration. Intracellular IL-4 staining indicated that 92% of IL-4 producing cells were consumed by 19A2 anti-CRTh2 antibody treatment, while IFNg producing cells were not reduced (Fig. 9A).

Anti-CRTh2 depletes congenital type 2 cells in human CRTh2.Bac.Tg mice

To assess the ability of anti-CRTh2 antibodies to consume congenital type 2 (IT2) cells (also known as congenital lymphoid 2 cells or ILC2 cells), the plasmid containing IL-17E was injected into hCRTh2.Bac.Tg mice. Increase the number of IT2 cells. Mice were treated with a single dose of anti-hCRTh2 or isotype control antibody i.v. and the percentage and number of IT2 cells in the mesenteric lymph nodes were measured by flow cytometry on day 3 post-treatment. Anti-hCRTh2 treatment The percentage and number of IT2 cells in mesenteric lymph nodes in hCRTh2.Bac.Tg mice were significantly reduced by more than 50%.

Example 2. Antibody humanization and affinity maturation Biotinylated CRTh2 expression in mammalian cells

Human, cynomolgus and cynomolgus CRTh2 cDNA and human CRTh2 with Q16E or R19H mutation were cloned into a mammalian expression vector fused in the framework at the 3' end to the encoded sequence and linker and the Avitag sequence (GSGGLNDIFEAQKIEWH) In pRK5. The Bir A biotin ligase gene from Escherichia coli was also cloned in the mammalian expression vector pRK5. Plasmids encoding CRTh2 from each species were mixed with BirA expression plasmid at a ratio of 9:1 and using Lipofectamine 2000 reagent (Invitrogen) in Dubec's modified Eagle's medium supplemented with 10% fetal bovine serum supplemented with 10 μM biotin. Transfected into 293T cells. After transfection, the cells were starved for 24 hours and the plasma membrane fraction containing biotinylated CRTh2 was purified.

Purification of plasma membrane fraction

Transfected cells (2.5 x 10 ⁸ ) were washed twice in PBS containing protease inhibitor cocktail (Roche) (150 mM NaCl, 10 mM sodium phosphate, pH 7.4) and the cell pellet was frozen at -80 °C. The cells were thawed and resuspended in 4 ml lysis buffer (1 mM EDTA, 50 mM HEPES buffer, pH 7.4, containing protease inhibitor cocktail) and lysed in 8 Dats using a tight fit in a Dounce homogenizer. . After the initial lysis, 4 ml of lysis buffer containing 500 mM sucrose was added and further lysed with 8 taps using a tight fit. Cell debris was removed by centrifugation at 770 x g for 10 minutes and the membrane material in the supernatant was precipitated by centrifugation at 17,000 x g . The precipitated membrane was resuspended in 6 ml lysis buffer containing 250 mM sucrose using 8 beats of loose fit in a Dounce homogenizer. Large debris was removed by centrifugation at 770 x g for 10 minutes. The supernatant was carefully plated onto 4 ml of lysis buffer containing 1.12 M sucrose in a transparent SW40 centrifuge tube and spun at 25,000 rpm for 1 hour in a SW40Ti rotator (Beckman) at 25,000 rpm. The material at the interface between the high and low concentrations of sucrose fraction was collected by pipette, mixed with an equal volume of sucrose-free lysis buffer and pelleted by centrifugation at 16,000 x g for 10 minutes at 4 °C. The precipitated plasma membrane was resuspended in 1 ml of lysis buffer and stored at -80 °C. All homogenization steps were performed on ice.

Performance and purification of CRTh2 from insect cells

Protein expression: The DNA encoding Ala3-Asp330 encoding Homo sapiens and Macaca fascicularis CRTh2 was cloned into a modified pAcGP67 baculovirus transfer vector containing a C-terminal Avi-tag and a His8-tag. In Biosciences). Recombinant baculoviruses were generated by co-transfection of Sf9 cells with the pacGP67 construct and linearized baculovirus DNA in ESF 921 medium (Expression Systems) using the BaculoGold Expression System (BD Biosciences). The virus was generated by three rounds of amplification. A recombinant baculovirus expressing unlabeled Escherichia coli BirA (Met1-Lys321) was generated in a similar manner. 10 L of Tni.PRO cells were co-infected with 40 mL of both viruses (CRTh2 and BirA) at a density of 2 x 10 ⁶ cells/mL. The cells were further grown for 48 hours at 27 ° C and removed from the culture medium by centrifugation.

Protein purification: The harvested cell pellet was resuspended in 50 mM Tris pH 8, 200 mM NaCl (TBS) containing a completely EDTA-free protease inhibitor cocktail (Roche) and lysed by three passes through a microfluidizer. After the lysate was clarified, the membrane was harvested by centrifugation at 40 K in a 45 Ti ultracentrifugal rotator (Beckman) at 4 °C for 2 hours. 30 g of the membrane precipitate was resuspended in TBS (10 g/L) and dissolved with 1% (wt/vol) lauryl maltose neopentyl glycol (LMNG, Affymetrix) at 4 ° C for 2 hours. After clarification, the samples were batch-bound to Ni-NTA resin (Qiagen) and washed with TBS containing 15 mM imidazole containing 0.12% (wt/vol) digitonin (EMD Biosciences). The protein was eluted with the same buffer containing 300 mM imidazole, concentrated and diluted in an imidazole-free TBS-gergerin (0.12%) buffer at 4 °C using a 100 MWKO rotary concentrator (Vivaspin, GE Healthcare). Times. Estimating biotinylation by comparison with protein standards - CRTh2 protein concentration; the sample was divided into aliquots and snap frozen in liquid nitrogen.

ELISA using solubilized CRTh2

Neutravidin (Pierce) was applied to a 96-well Maxisorp ELISA plate (2 μg/ml in 10 mM carbonate buffer, pH 9.6, 100 μl/well) overnight at 4 ° C with 0.5% bovine serum Albumin in PBS (blocking buffer) was blocked. The plasma membrane containing CRTh2 or control membrane protein or purified CRTh2 was diluted in blocking buffer and lysed on ice in 1% dodecyl maltoside (DDM) for 15 minutes and at 16,000 x g The insoluble matter was removed by centrifugation at 4 ° C for 30 minutes. The solubilized CRTh2 or control membrane protein was diluted in blocking buffer containing 0.2% DDM and added to a neutravidin coated plate. The proteins were incubated for 10 minutes and the plates were washed with PBS containing 0.05% DDM, and the antibodies were serially diluted in blocking buffer containing 0.2% DDM and incubated with the captured antigen for 1 hour at 4 °C. The plates were washed as described above and the anti-human or anti-mouse IgG conjugated to peroxidase diluted in 0.2% DDM in capping buffer was added to the plates. After incubation at 4 ° C for 30 minutes, the plates were washed as described above and the TMB substrate was added to the plates. The peroxidase reaction was terminated with an equal volume of 1 M phosphoric acid and the optical absorbance was read at 450 nm. The amount of CRTh2 protein used is sufficient to achieve saturation of the wells, as determined by ELISA using anti-CRTh2 Mab binding to recombinant human, cynomolgus and rhesus CRTh2.

Antibody humanization

The CDR sequences of Mab 19A2 (Figure 10) were grafted onto the consensus kappa 1 (common K1) and consensus VH3 (common H3) frameworks by oligonucleotide-directed site-directed mutagenesis (Dennis, MS (2010). CDR repair : A novel approach to antibody humanization. in Current Trends in Monoclonal Development and Manufacturing, SJ Shire, W. Gombotz, K. Bechtold-Peters and J. Andya, (Springer, New York), pp. 9-28). The 71-position (Kabat numbering system) of the light chain present in the parental murine 19A2 antibody and the framework residue at position 49 of the heavy chain were also incorporated into the framework position of the humanized antibody hu19A2.v1 (Figs. 11A and 11B). . Guided by oligonucleotides The CDR sequences of Mab 8B1 (Figure 12) were grafted onto the consensus K1 and consensus VH1 (shared H1) frameworks. Also included in the parental murine 8B1 antibody are the 46, 66, 69 and 71 positions of the light chain and the framework residues of 37, 67, 69, 71 and 91 of the heavy chain. The position of the antibody hu8B1.v1 is in the frame position (Fig. 12). All antibodies were colonized in the pRK5 vector. The humanized antibody appeared as human IgG1 in 293T cells and purified by affinity chromatography on protein A-Sepharose. The binding of Mab was determined by ELISA using human, cynomolgus and rhesus monkey CRTh2.

Affinity maturity

The heavy and light chain variable regions of hu19A2.v1 were cloned into a phage display vector displaying a Fab fragment fused to the p3 protein of bacteriophage M13. Two sets of "terminator" template vectors were prepared in which all three CDRs of the light or heavy chain were removed and replaced with sequences encoding the stop codon. Libraries with randomized heavy or light chain CDRs were generated by oligonucleotide directed directed mutagenesis. Oligonucleotides directed against CDRs are synthesized, wherein each oligonucleotide has a codon that is randomized to NNK (N=A, T, C or G; K=T or G). The Kabat positions of the light chain were randomized to 27 to 34, 50 to 56 and 89 to 97 using a set of 24 oligonucleotides and the Kabat positions were 26 to 35, 49 to 58 and 95 using a set of 28 oligonucleotides. Randomized to 100a. The randomized library was electroporated in E. coli XL1-Blue cells (Agilent technologies), and the mutant-assisted phage KO7+ (Lamboy et al., ChemBioChem 9: 2846-2852 (2008)) was used to infect 10 cells/well. The infection was repeated, allowed to cover and grown overnight in 2YT broth containing 50 μg/ml carbenicillin and 100 μg/ml kanamycin at 37 °C. The cells were removed by centrifugation and the phage display Fabn in the supernatant was concentrated and purified by PEG precipitation (ref). The phage was submitted to 4 rounds of selection. In each round, phage in blocking buffer containing 0.2% DDM was solubilized at 4 °C with DDM with wild-type sequence or ELISA plate with Q16E or R19H mutation and binding to neutral avidin coating. The human CRTh2 was incubated for 1 hour. The plate was washed with PBS containing 0.05% DDM and the phage It was eluted with 100 μl of 0.1 N HCl for 10 minutes. Phage were collected and neutralized by the addition of 1/8 volume of 1 M Tris base. Phage were used to infect E. coli XL1-Blue and propagate as described above. Escherichia coli XL1-Blue was infected with phage from the fourth round and plated on LB containing 50 μg/ml carbenicillin to obtain an isolated pure line. The pure lines were sequenced by the double deoxygen chain terminator method and the mutations in each position were tabulated. A favorable mutation was introduced into the humanized hu19A2.v1 human IgG1 pure line and the IgG was expressed in human 293T cells and purified by affinity chromatography. The binding of IgG to human, rhesus and cynomolgus CRTh2 was tested by ELISA. A second generation library was generated based on hu19A2.v12 including the light chain mutation S31W and the heavy chain mutation Y58D. This second generation library was selected as described above, except that in selection, purified human and cynomolgus CRTh2 antigen lines were expressed in Sf9 cells and replaced with DMD by 0.12% digitonin. In addition, position 31 of the heavy chain is randomized with two oligonucleotides having degenerate codons NHK and VNK at the position, which are combined to encode all amines except tryptophan and cysteine. Base acid. The tryptophan acid at position 31 of the light chain in humanized 19A2.v58, 19A2.v60 and 19A2.v52 was changed to tyrosine. The sequence of 19A2.v46 is identical to 19A2.52 except that it contains tryptophan rather than tyrosine at position 31. The aspartic acid at position 56 of 19A2.v60 and other pure lines was changed to glutamic acid to remove the isomerization site.

In vitro characterization of mouse and humanized anti-human CRTh2 antibody generators

The reactivity of humanized lines h19A2.v1, h19A2.v46 and h19A2.v52 with human CRTh2 expressed on 293 cells was tested. Similar to 19A2, the humanized anti-CRTh2 antibody reacted with human CRTh2 expressed on 293 cells in a dose-dependent manner (Fig. 15A). In addition, humanized affinity matured lines h19A2.v12, h19A2.v46 and h19A2.v52 also showed dose-dependent reactivity with cynomolgus monkeys and rhesus monkey CRTh2 on 293 cells, whereas humanized antibody h19A2.v1 showed Cynomolgus and rhesus rhesus CRTh2 expressed on 293 cells were not reactive (Fig. 15B). In addition, the humanized and affinity matured antibody h19A2.v52 was dosed with primary humans from the whole blood, cynomolgus monkeys and rhesus monkeys with basophilic balls and eosinophils. Dependent mode response (Figure 15C).

Ligand analysis of radiolabels was performed using homologous competition to assess the dissociation constant (KD) of human and cynomolgus CRTh2 against the surface manifestations of anti-CRTh2 antibodies on 293 cells. To obtain direct measurements of the KD values of the humanized antibodies h19A2.v52 and h19A2.v46, Fab fragments of these antibodies were generated (Fig. 16). Humanized anti-CRTh2 pure lines 19A2.v52 and 19A2.v46 (Fab fragments of IgG) had KD values of 13.7 nM and 6.4 nM for 293 cells expressing human CRTh2, respectively. The KD values of h19A2.v52 and h19A2.v46 (Fab fragment of IgG) against 293 cells expressing cynomolgus CRTh2 were 21.3 nM and 8.6 nM, respectively. Based on these measurements, humans had a relative binding affinity for cynomolgus CRTh2 for h19A2.52 within 2 fold (Figures 16A and B) and appeared to be equivalent for h19A2.v46 (Figures 16C and D).

To assess the blocking function of humanized and affinity matured anti-CRTh2 antibodies, the effect of anti-CRTh2 antibodies on PGD2-mediated inhibition of forskolin-induced cAMP levels in 293 cells expressing human CRTh2 was tested. Forskolin plus PGD2-stimulated hCRTh2 expression 293 cells treated with 8B1 antibody increased cAMP content in a dose-dependent manner (Fig. 17A). Thus, the 8B1 antibody blocked the PGD2-mediated decrease in forskolin-induced cAMP content in a dose-dependent manner, indicating that 8B1 has a PGD2 function blocking ability similar to its ability to inhibit calcium flux in Th2 cells in response to PGD2. In contrast, h19A2.v52 did not show PGD2 function blocking ability in forskolin-induced cAMP human CRTh2 293 cell assay. It was also tested whether anti-CRTh2 antibodies have a direct effect on forskolin-induced cAMP levels in the absence of PGD2. As shown in Figure 17B, various concentrations of anti-CRTh2 antibodies did not show an effect on forskolin-induced cAMP content in human CRTh2 293 cells, indicating that these antibodies did not exhibit agonistic activity in this assay. In contrast, PGD2 reduced forskolin-induced cAMP levels.

To test whether anti-CRTh2 antibody 19A2 can deplete CRTh2+ basophilic and eosinophils in blood, spleen and bone marrow in vivo, a single dose of 19A2 antibody is administered to CRTh2.Bac.Tg mice, as indicated (Figure 18A and B). Single dose of 20ug/mouse or 19A2 of 100ug/mouse completely consumed basophilic globules and eosinophils in peripheral blood and spleen of human CRTh2.Bac.Tg mice on the third day after treatment, and consumed blood and spleen on the 7th day after treatment. Eosinophilic spheres in the bone marrow, as determined by flow cytometry (Figures 18A, B and C). Significant consumption of basophilic bulbs was still observed in the spleen at two doses on day 7 post-treatment, while basophilic bulb consumption in the bone marrow was more pronounced with a dose of 100 ug/mouse. The consumption of basophilic balls in the blood varied on the 7th day after treatment.

To test whether the humanized and affinity matured anti-CRTh2 antibody h19A2.v52 can consume CRTh2+ basophilic globules and eosinophils in blood, spleen and bone marrow in vivo, a single dose of 0.5mg/kg or 10mg/kg 19A2. The v52 hIgG1 antibody was administered to human CRTh2.Bac.Tg mice (Fig. 19A). A single dose of 0.5 mg/kg or 10 mg/kg of h19A2.v52 on day 2 completely consumed eosinophils in peripheral blood, spleen or bone marrow in human CRTh2.Bac.Tg mice as determined by flow cytometry. On days 7 and 14, the spleen (Fig. 19B) and bone marrow (Fig. 19C) and eosinophilic spheres in the blood (Fig. 19A) were still consumed at the 10 mg/kg dose. In contrast, at the dose of 0.5 mg/kg, the eosinophilic spheres in the blood, spleen and bone marrow were partially returned to baseline on day 7 and partially on day 14. In addition, significant consumption of basophilic bulbs was observed in the spleen on day 2 at two doses and on day 7 at a dose of 10 mg/kg, while basophilic bulb consumption in the bone marrow utilized two doses. Days 2 and 7 were less obvious. The basophilic ball content in the spleen returned to baseline at day 5 at a dose of 0.5 mg/kg and returned to baseline in both spleen and bone marrow on day 14 at both doses.

To assess whether the effector function of anti-CRTh2 antibodies is required for adequate consumption of CRTh2+ cells in tissues, in hCRTh2.Bac.Tg mice in congenital immune cell depletion and Th2 BAL cells in a TNP-OVA-induced chronic asthma model The reduction in hormonal production was compared to the Fc mutant anti-CRTh2 19A2_DANA antibody and anti-CRTh2 19A2. In the ip200ug/mouse regimen, four doses of antibody 19A2 consumed 96%, 86%, and 72% of lung eosinophils, basophils, and mast cells, respectively (Fig. 20A); 19A2_DANA Treatment only partially reduced eosinophils, basophils, and mast cells in the lung by 26%, 34%, and 31%, respectively (Fig. 20A). In addition, treatment with 19A2 consumed 99% of bronchoalveolar lavage (BAL) fluid eosinophils, but only 19% with 19A2_DANA treatment. Furthermore, the Th2 cytokine IL-4 in BAL fluid was reduced by 100% after 19A2 treatment, whereas 19A2_DANA treatment resulted in only a 20% reduction in BAL IL-4 (Fig. 16B).

Murine antibody variable sequence

mu19A2-light chain variable region (SEQ ID NO: 49)

mu19A2-heavy chain variable region (SEQ ID NO: 61)

mu8B1-light chain variable region (SEQ ID NO: 50)

mu8B1-heavy chain variable region (SEQ ID NO: 62)

mu3C12-light chain variable region (SEQ ID NO: 51)

mu3C12-heavy chain variable region (SEQ ID NO: 63)

mu31A5-light chain variable region (SEQ ID NO: 53)

mu31A5-heavy chain variable region (SEQ ID NO: 65)

Humanized 8B1 variable region sequence

hu8B1.v1-light chain variable region (SEQ ID NO: 52)

hu8B1.v1-heavy chain variable region (SEQ ID NO: 64)

Humanized 19A2 variable region sequence

hu19A2.v1-light chain variable region (SEQ ID NO: 38)

hu19A2.v12-light chain variable region (SEQ ID NO: 39)

hu19A2.v38-light chain variable region (SEQ ID NO: 39)

hu19A2.v46-light chain variable region (SEQ ID NO: 39)

hu19A2.v47-light chain variable region (SEQ ID NO: 39)

hu19A2.v51-light chain variable region (SEQ ID NO: 39)

hu19A2.v52-light chain variable region (SEQ ID NO: 40)

hu19A2.v53-light chain variable region (SEQ ID NO: 40)

hu19A2.v57-light chain variable region (SEQ ID NO: 41)

hu19A2.v58-light chain variable region (SEQ ID NO: 42)

hu19A2.v60-light chain variable region (SEQ ID NO: 41)

hu19A2.v61-light chain variable region (SEQ ID NO: 42)

hu19A2.v62-light chain variable region (SEQ ID NO: 41)

hu19A2.v63-light chain variable region (SEQ ID NO: 43)

hu19A2.v64-light chain variable region (SEQ ID NO: 42)

hu19A2.v65-light chain variable region (SEQ ID NO: 43)

hu19A2.v66-light chain variable region (SEQ ID NO: 44)

hu19A2.v67-light chain variable region (SEQ ID NO: 45)

hu19A2.v68-light chain variable region (SEQ ID NO: 44)

hu19A2.v69-light chain variable region (SEQ ID NO: 45)

hu19A2.v70-light chain variable region (SEQ ID NO: 46)

hu19A2.v71-light chain variable region (SEQ ID NO: 47)

hu19A2.v72-light chain variable region (SEQ ID NO: 48)

hu19A2.v1-heavy chain variable region (SEQ ID NO: 54)

hu19A2.v12-heavy chain variable region (SEQ ID NO: 55)

hu19A2.v38-heavy chain variable region (SEQ ID NO: 56)

hu19A2.v46-heavy chain variable region (SEQ ID NO: 57)

hu19A2.v47-heavy chain variable region (SEQ ID NO: 58)

hu19A2.vS1-heavy chain variable region (SEQ ID NO: 59)

hu19A2.v52-heavy chain variable region (SEQ ID NO: 57)

hu19A2.v53-heavy chain variable region (SEQ ID NO: 59)

hu19A2.v57-heavy chain variable region (SEQ ID NO: 59)

hu19A2.v58-heavy chain variable region (SEQ ID NO: 57)

hu19A2.v60-heavy chain variable region (SEQ ID NO: 57)

hu19A2.v61-heavy chain variable region (SEQ ID NO: 55)

hu19A2.v62-heavy chain variable region (SEQ ID NO: 55)

hu19A2.v63-heavy chain variable region (SEQ ID NO: 55)

hu19A2.v64-heavy chain variable region (SEQ ID NO: 60)

hu19A2.v65-heavy chain variable region (SEQ ID NO: 60)

hu19A2.v66-heavy chain variable region (SEQ ID NO: 55)

hu19A2.v67-heavy chain variable region (SEQ ID NO: 55)

hu19A2.v68 (SEQ ID NO: 60)

hu19A2.v69 (SEQ ID NO: 60)

hu19A2.v70-heavy chain variable region (SEQ ID NO: 54)

hu19A2.v71-heavy chain variable region (SEQ ID NO: 54)

hu19A2.v72-heavy chain variable region (SEQ ID NO: 54)

Humanized 19A2 full length sequence

hu19A2.v1-light chain (SEQ ID NO: 66)

hu19A2.v12-light chain (SEQ ID NO: 67)

hu19A2.v38-light chain (SEQ ID NO: 67)

hu19A2.v46-light chain (SEQ ID NO: 67)

hu19A2.v47-light chain (SEQ ID NO: 67)

hu19A2.v51-light chain (SEQ ID NO: 67)

hu19A2.v52-light chain (SEQ ID NO: 68)

hu19A2.v53-light chain (SEQ ID NO: 68)

hu19A2.v57-light chain (SEQ ID NO: 69)

hu19A2.v58-light chain (SEQ ID NO: 70)

hu19A2.v60-light chain (SEQ ID NO: 69)

hu19A2.v61-light chain (SEQ ID NO: 70)

hu19A2.v62-light chain (SEQ ID NO: 69)

hu19A2.v63-light chain (SEQ ID NO: 71)

hu19A2.v64-light chain (SEQ ID NO: 70)

hu19A2.v65-light chain (SEQ ID NO: 71)

hu19A2.v66-light chain (SEQ ID NO: 72)

hu19A2.v67-light chain (SEQ ID NO: 73)

hu19A2.v68-light chain (SEQ ID NO: 72)

hu19A2.v69-light chain (SEQ ID NO: 73)

hu19A2.v70-light chain (SEQ ID NO: 74)

hu19A2.v71-light chain (SEQ ID NO: 75)

hu19A2.v72-light chain (SEQ ID NO: 76)

hu19A2.v1-heavy chain (SEQ ID NO: 77)

hu19A2.v12-heavy chain (SEQ ID NO: 78)

hu19A2.v38-heavy chain (SEQ ID NO: 79)

hu19A2.v46-heavy chain (SEQ ID NO: 80)

hu19A2.v47-heavy chain (SEQ ID NO: 81)

hu19A2.v51-heavy chain (SEQ ID NO: 82)

hu19A2.v52-heavy chain (SEQ ID NO: 80)

hu19A2.v53-heavy chain (SEQ ID NO: 82)

hu19A2.v57-heavy chain (SEQ ID NO: 82)

hu19A2.v58-heavy chain (SEQ ID NO: 80)

hu19A2.v60-heavy chain (SEQ ID NO: 80)

hu19A2.v61-heavy chain (SEQ ID NO: 78)

hu19A2.v62-heavy chain (SEQ ID NO: 78)

hu19A2.v63-heavy chain (SEQ ID NO: 78)

hu19A2.v64-heavy chain (SEQ ID NO: 83)

hu19A2.v65-heavy chain (SEQ ID NO: 83)

hu19A2.v66-heavy chain (SEQ ID NO: 78)

hu19A2.v67-heavy chain (SEQ ID NO: 78)

hu19A2.v68-heavy chain (SEQ ID NO: 83)

hu19A2.v69-heavy chain (SEQ ID NO: 83)

hu19A2.v70-heavy chain (SEQ ID NO: 77)

hu19A2.v71-heavy chain (SEQ ID NO: 77)

hu19A2.v72-heavy chain (SEQ ID NO: 77)

Periosteal protein sequence

Human periostin isoform 1 NP_006466 (SEQ ID NO: 87)

Human periostin isoform 2 NP_001129406 (SEQ ID NO: 88)

Human periostin isoform 3 NP_001129407 (SEQ ID NO: 89)

Human periostin isoform 4 NP_001129408 (SEQ ID NO: 90)

Human periostin isoform 5 (SEQ ID NO: 91)

The invention has been described in considerable detail by way of illustration and example, and the description and examples should not be construed as limiting the scope of the invention. this The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety.

<110> Kailin Ruifu Isidro Hernil Joanne S Hongge Tao Huang Yong Lei Miredis Hessen

<120> Anti-CRTH2 antibody and method of use

<130> 146392017340

<140> Not Yet Assigned

<141> Concurrently Herewith

<150> US 61/786,370

<151> 2013-03-15

<160> 91

<170> FastSEQ for Windows Version 4.0

<210> 1

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<400> 91

Claims (72)

An isolated antibody that binds to human CRTh2 and consumes CRTh2 expressing cells when administered to a human subject in a therapeutically effective amount. The antibody of claim 1, wherein the antibody consumes one or more of the following types of CRTh2 expressing cells: Th2 cells, mast cells, eosinophils, basophilic spheres or congenital type 2 (IT2) cells. The antibody of claim 1 or 2, wherein the antibody has been engineered to improve ADCC and/or CDC activity. The antibody of claim 1 or 2, wherein the antibody has been engineered to improve ADCC and/or to reduce CDC activity. The antibody of any one of claims 1 to 4, wherein the antibody is afucosylated. The antibody of claim 5, wherein the anti-system is produced in a cell line having an α1,6-fucosyltransferase (Fut8) knockout. The antibody of claim 5, wherein the anti-system is produced in a cell line that overexpresses β1,4- N -ethinylglucosamine transferase III (GnT-III). The antibody of claim 7, wherein the cell line additionally exhibits Golgi alpha-mannosidase II (ManII). The antibody of claim 3, wherein the antibody comprises at least one amino acid substitution in the Fc region that improves ADCC and/or CDC activity. The antibody of claim 9, wherein the amino acid is substituted by the system S298A/E333A/K334A. The antibody of any one of claims 1 to 10, wherein the anti-system naked antibody. The antibody of any one of claims 1 to 11, wherein the anti-system chimeric antibody. The antibody of any one of claims 1 to 11, wherein the anti-system humanized antibody. The antibody of any one of claims 1 to 11, wherein the anti-system human antibody. The antibody of any one of claims 1 to 14, wherein the anti-system bispecific antibody. The antibody of any one of claims 1 to 14, wherein the anti-system IgG1 antibody. The antibody of any one of claims 1 to 16, wherein the antibody competitively inhibits binding of at least one of the following antibodies to human CRTh2: 19A2, 8B1, 31A5, and 3C12. The antibody of claim 17, wherein an ELISA assay is used to determine competitive binding. The antibody of any one of claims 1 to 18, wherein the antibody binds to an epitope of human CRTh2 that is identical to or overlaps with a CRTh2 epitope that binds to at least one of the following anti-CRTh2 antibodies: 19A2, 8B1, 31A5 And 3C12. The antibody of any one of claims 1 to 19, wherein the antibody comprises six hypervariable regions (HVR) from one of the following anti-CRTh2 antibodies: 19A2, 8B1, 31A5 and 3C12. The antibody of any one of claims 1 to 16, wherein the antibody binds to CRTh2 of a non-human primate. The antibody of claim 21, wherein the antibody binds to rhesus monkey and/or cynomolgus CRTh2. The antibody of any one of claims 1 to 22, wherein the antibody further blocks CRTh2 signaling. The antibody of any one of claims 1 to 22, wherein the antibody prevents CRTh2 expressing cells from responding to recruitment of prostaglandin D2. The antibody of any one of claims 1 to 22, wherein the antibody blocks Ca2 ⁺ flux in CRTh2 expressing cells. The antibody of any one of claims 1 to 25, wherein the antibody binds to human CRTh2 with a Kd value of about 100 nM or less. The antibody of claim 1, wherein the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and X ₁ ISNGGSTTX ₂ YPGTVEG ( HVR-H2 of SEQ ID NO: 5), wherein X ₁ is Y or R, and X ₂ is Y or D. The antibody of claim 1, wherein the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35 or 36, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and comprising SEQ ID NO: HVR-H2 of the amino acid sequence of 32 or 33. An isolated anti-CRTh2 antibody comprising a light chain and a heavy chain variable region, wherein the light and heavy chain variable region comprises six hypervariable region (HVR) sequences: (i) HVR-L1 comprising RASENIYXNLA (SEQ ID NO: 1), wherein X is S, W or Y; (ii) HVR-L2 comprising AATQLAX (SEQ ID NO: 2), wherein X is D, E or S; (iii) HVR-L3 , comprising QHFWITPWT (SEQ ID NO: 3); (iv) HVR-H1 comprising X ₁ YX ₂ MS (SEQ ID NO: 4), wherein X ₁ is S or F, and X ₂ is S, L or K; (v) HVR-H2 comprising X ₁ ISNGGSTTX ₂ YPGTVEG (SEQ ID NO: 5), wherein X ₁ is Y or R, and X ₂ is Y or D; and (vi) HVR-H3, which comprises HRTNWDFDY (SEQ ID NO: 6). An isolated anti-CRTh2 antibody comprising a light chain and a heavy chain variable region, wherein the light chain variable region comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7, 8, or 9, comprising SEQ ID NO: HVR-L2 of the amino acid sequence of 10, 11 or 12 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3. The antibody of claim 29, which further comprises the heavy chain variable region comprising: HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16 or 17, comprising SEQ ID NO: 18 HVR-H2 of the amino acid sequence of 19, 20 or 21 and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6. An isolated anti-CRTh2 antibody comprising a light chain and a heavy chain variable region, comprising The heavy chain variable region comprising: HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16 or 17 and an amino acid comprising SEQ ID NO: 18, 19, 20 or 21. Sequence HVR-H2 and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6. The antibody of any one of claims 29 to 32, wherein the antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-L2 comprising SEQ ID NO : an amino acid sequence of 10; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6. The antibody of any one of claims 29 to 32, wherein the antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (ii) HVR-L2 comprising SEQ ID NO : an amino acid sequence of 10; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 19; and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6. The antibody of any one of claims 29 to 32, wherein the antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-L2 comprising SEQ ID NO An amino acid sequence of 11; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6. An isolated anti-CRTh2 antibody comprising a light chain and a heavy chain variable region, wherein The antibody comprises a VL sequence selected from the group consisting of SEQ ID NOs: 38-53. The antibody of claim 36, wherein the antibody further comprises a VH sequence selected from the group consisting of SEQ ID NOs: 54-65. An isolated anti-CRTh2 antibody comprising a light chain and a heavy chain variable region, wherein the antibody comprises a VH sequence selected from the group consisting of SEQ ID NOs: 54-65. The antibody of any one of claims 36 to 38, which comprises the VL sequence of SEQ ID NO: 40 and the VH sequence of SEQ ID NO: 57. The antibody of any one of claims 36 to 38, which comprises the VL sequence of SEQ ID NO: 39 and the VH sequence of SEQ ID NO: 55. The antibody of any one of claims 36 to 38, wherein the antibody comprises the VL sequence of SEQ ID NO: 41 and the VH sequence of SEQ ID NO: 57. The antibody of any one of claims 29 to 41, wherein the anti-system monoclonal antibody. The antibody of any one of claims 29 to 41, wherein the anti-system is a humanized or chimeric antibody. The antibody of any one of claims 29 to 41, wherein at least a portion of the framework sequence is a human consensus framework sequence. The antibody of any one of claims 29 to 44, wherein the anti-system is selected from the group consisting of Fab, Fab'-SH, Fv, scFc or (Fab') ₂ fragments. An antigen-binding fragment of an antibody according to any one of claims 1 to 44. An isolated nucleic acid encoding the antibody of any one of claims 1 to 45 and the antigen-binding fragment of claim 46. A host cell comprising the nucleic acid of claim 47. A method of producing an antibody comprising culturing a host cell as claimed in claim 48 such that the antibody is produced. The method of claim 49, further comprising recovering the antibody produced by the host cell. An immunoconjugate comprising the antibody of any one of claims 1 to 45 or the antigen-binding fragment of claim 46 and a cytotoxic agent. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 45 or an antigen-binding fragment of claim 46 and a pharmaceutically acceptable carrier. A method of treating asthma comprising administering an effective amount of an anti-CRTh2 antibody to an individual, wherein the antibody consumes CRTh2 expressing cells in the individual. The method of claim 53, wherein the antibody consumes one or more of the following types of CRTh2 expressing cells: Th2 cells, mast cells, eosinophils, basophilic spheres or congenital type 2 (IT2) cells. The method of claim 53 or 54, wherein the anti-CRTh2 antibody consumes CRTh2 expressing cells from lung tissue. The method of any one of claims 53 to 55, wherein the anti-CRTh2 antibody consumes CRTh2 expressing cells from bronchoalveolar lavage fluid. The method of any one of claims 53 to 55, wherein the anti-CRTh2 antibody consumes at least 50% of at least one type of CRTh2 expressing cells from the lung compared to the baseline prior to administration of the antibody. The method of claim 57, wherein the anti-CRTh2 antibody consumes at least 80% of at least one type of CRTh2 expressing cells from the lung compared to the baseline prior to administration of the antibody. The method of claim 57, wherein the anti-CRTh2 antibody consumes at least 90% of at least one type of CRTh2 expressing cells from the lung compared to the baseline prior to administration of the antibody. The method of any one of claims 53 to 59, wherein the individual has granule-poll-type asthma. The method of any one of claims 53 to 60, wherein the content of one or more cytokines in the individual is decreased after administration of the anti-CRTh2 antibody. The method of claim 61, wherein the content of one or more cytokines produced by at least one of the following cell types is decreased: Th2 cells, mast cells, eosinophils, basophils or congenital type 2 (IT2) )cell. The method of claim 61, wherein the individual has a reduced content of one or more of IL-4, IL-5, IL-9, IL-13, IL-17, histamine or leukotriene. The method of any one of claims 53 to 59, wherein the individual has an inhaled corticosteroid, a short acting beta 2 agonist, a long acting beta 2 agonist or a combination thereof to control insufficient asthma. The method of any one of clauses 53 to 64, wherein the system is human. The method of any one of claims 53 to 65, wherein the anti-CRTh2 anti-system is an antibody of any one of claims 1 to 45 or an antigen-binding fragment of claim 46. A method of treating a condition mediated by a CRTh2 expressing cell comprising administering an effective amount of an anti-CRTh2 antibody to an individual, wherein the antibody depletes a CRTh2 expressing cell in the individual. The method of claim 67, wherein the condition is selected from the group consisting of asthma, granule-deficient asthma, atopic dermatitis, allergic rhinitis, acute or chronic airway hypersensitivity, eosinophilic syndrome, hobby Acidic globular esophagitis, Churg-Strauss syndrome, idiopathic pulmonary fibrosis, inflammation associated with cytokines, inflammation associated with CRTh2 expressing cells, associated with CRTh2 expressing cells Malignant neoplasms, chronic idiopathic urticaria, chronic spontaneous urticaria, physical urticaria, cold urticaria, stress urticaria, bullous pemphigoid, nasal sputum, food allergies and allergic bronchopulmonary Ascariasis (ABPA). The method of claim 67 or 68, wherein the anti-CRTh2 anti-system is an antibody of any one of claims 1 to 45 or an antigen-binding fragment of claim 46. A method of reducing the content of a cytokine in an individual, comprising an effective amount of resistance The CRTh2 antibody is administered to the individual, wherein the antibody depletes CRTh2 expressing cells in the individual. The method of claim 70, wherein the individual has a reduced content of one or more of IL-4, IL-5, IL-9, IL-13, IL-17, histamine or leukotriene. The method of claim 70 or 71, wherein the anti-CRTh2 anti-system is an antibody of any one of claims 1 to 45 or an antigen-binding fragment of claim 46.